Journal: Scientific Reports

Article Title: TRIM39 aggravates hepatocellular carcinoma growth through targeting the p62-KEAP1-NRF2 axis

doi: 10.1038/s41598-025-32976-x

Figure Lengend Snippet: p62 is a binding partner of TRIM39. ( A ) Cell lysates from HepG2 cells transfected with empty vector or Flag-TRIM39 were immunoprecipitated with anti-Flag antibody. p62 was identified via mass spectrometry. ( B ) HepG2 cells were co-transfected with Flag-TRIM39 and Myc-p62 for 48 h. Total cell lysates were immunoprecipitated with anti-Flag or anti-Myc antibodies. Myc-p62 and Flag-TRIM39 were detected by western blot. ( C-D ) Immunofluorescence assay of Flag-TRIM39 and Myc-p62 in HepG2 cells. Representative confocal microscopy images were shown. Scale bars, 5 μm. The colocalization of TRIM39 and p62 in HepG2 cells was analyzed. ( E ) The analysis of p62 mRNA levels in TRIM39 stably overexpression or knockdown HCC cells. ( F - G ) The analysis of p62 protein levels in TRIM39 stably overexpression HepG2 and Huh7 cells. *, P < 0.05. ( H - I ) The analysis of p62 protein levels in TRIM39 stably knockdown MHCC97 and HCCLM3 cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001. ( J ) HepG2 cells transfected with Flag-TRIM39 were treated with cycloheximide (CHX, 10µM) for indicated time points and subjected to western blot analysis. The quantitative result of p62 protein level was analyzed. ( K ) TRIM39 stably knockdown MHCC97 cells were treated with cycloheximide (CHX, 10µM) for indicated time points and subjected to western blot analysis. The quantitative result of p62 protein level was analyzed. ( L ) TRIM39 stably knockdown MHCC97 cells were treated with cycloheximide (CHX, 10µM) and MG132 (10µM) for indicated time and subjected to western blot analysis. The quantitative result of p62 protein level was analyzed. ( M ) TRIM39 stably overexpression and control cell lysates were immunoprecipitated with anti-p62 antibody. Immunoprecipitation complex was detected by anti-p62 and anti-Ub antibodies. ( N ) TRIM39 stably knockdown and shNC cell lysates were immunoprecipitated with anti-p62 antibody. Immunoprecipitation complex was detected by anti-p62 and anti-Ub antibodies. ( O ) TRIM39 facilitated p62 Ub conjugates by in vitro ubiquitination assay. ( P ) TRIM39 stably overexpression cells were co-transfected with indicated HA-Ub (K-R) mutants for 48 h. Total lysates were immunoprecipitated with anti-p62 antibody. The immunoprecipitation complex was analyzed by western blot with anti-HA and anti-p62 antibodies. ( Q ) TRIM39 stably overexpression cells were transfected with Flag-p62 and its mutants (K420R, K435R). The expression of Flag-p62 was analyzed by western blot.

Article Snippet: Briefly, Purified recombinant His-p62 and GST-TRIM39 proteins were incubated in a mixture of ubiquitin ligation reaction containing 5 mM MgCl2, 50 mM Tris-HCl (pH 7.5), 2 mM DTT, 4 mM ATP, 1 μg ubiquitin (Yeasen, 20431ES08), 300 ng E1-UBA1 (Targetmol, TMPY-02840), and 500 ng E2-UBE2L3 (Targetmol, TMPY-02842) at 30 °C for 3 h. The reaction was terminated by boiling for 10 min in 2× SDS sample buffer, and the proteins were resolved by SDS-PAGE followed by western blotting with the indicated antibodies.

Techniques: Binding Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Mass Spectrometry, Western Blot, Immunofluorescence, Confocal, Microscopy, Analysis, Stable Transfection, Over Expression, Knockdown, Control, In Vitro, Ubiquitin Proteomics, Expressing, Co-Immunoprecipitation Assay

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: a, Schematic representation of the yeast two-hybrid screen performed using Gal4-DBD-Sam68 as bait and a Gal4-AD fusion cDNA library from LNCaP cells, b, Table reporting the Sam68-interacting factors identified by the screen, c, Five clones of the AH109 yeast strain transformed with the plasmid expressing Gal4-AD-XRN2 (1,929–2,842 nt) (clone 177) and Gal4-DBD-Sam68 fusion proteins, or both plasmids co-transformed with empty vectors as controls. Clones were plated in non-stringency (SD without Leu and Trp) and high-stringency (SD without Leu, Trp, His and Ade) medium and grown at 28 °C for four days, d, Scheme of the XRN2 structure with the position of the Sam68-interacting region (red box), e. Representative western-blot analysis of the reciprocal co-immunoprecipitation (co-IP) between endogenous Sam68 and XRN2 from LNCaP nuclear extracts using Sam68 (α-Sam68) or XRN2 (α-XRN2) antibodies (n = 3). Input = 0.25%. f, Representative western-blot analysis of the co-IP of endogenous Sam68 with XRN2, performed using LNCaP nuclear extracts (NE) in the presence (+) or absence (−) of RNaseA (n = 3). A representative agarose gel of RNA degradation is also shown (RNA). In e and f, non-immune rabbit immunoglobulins G (α-IgG) were used as a negative control.

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: Two Hybrid Screening, cDNA Library Assay, Clone Assay, Transformation Assay, Plasmid Preparation, Expressing, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Agarose Gel Electrophoresis, Negative Control

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: RNA Binding Assay

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: RNA Binding Assay

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polvadenylation to cell cycle progression in prostate cancer

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: RNA Binding Assay

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: RNA Binding Assay

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: a, Pearson’s correlation analyses of XRN2 and MYC expression in the PC Jenkins dataset (GSE46691). Pearson’s correlation coefficient (r; two-sided) and P value are reported (95% confidence interval), b, Dot plot showing the distribution of XRN2 expression in patients with PC (Jenkins dataset, GSE46691), classified into Sam68low (blue circles) and Sam68high (red squares) expression groups according to Z-score normalization. The median is shown as a solid horizontal line, c, Representative images of immunohistochemistry analyses of patients with PC (n = 20) with low and high expression of XRN2 and Sam68. Spearman’s correlation is reported (ρ = 0.653; P = 0.002). d, Violin plot showing the correlation between Sam68 and XRN2 expression with Gleason score, in patients with PC (Jenkins dataset, GSE46691). In b and d, statistical significance was calculated by the Mann-Whitney test (two-sided), and P values are reported (95% confidence interval).

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: Expressing, Immunohistochemistry, MANN-WHITNEY

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: RNA Binding Assay

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: a, Bar graph showing the percentage of 3’UTR- and CDS-APA events annotated in the genes expressed in LNCaP cells (white columns) and the percentage of those that are differentially regulated in Sam68- and XRN2-depleted cells (gray columns). Statistical significance wascalculated by modified Fisher’s exact test (two-sided, 95% confidence interval), and the exact P values are reported. b,c, Representative western-blot (b) and densitometric analyses (c) of subcellular fractionation experiments (n = 3) performed in control (sh-scr), Sam68 (sh-Sam68) and XRN2 (sh-XRN2) stably depleted LNCaP cells. CE, total cell extract; Cyt, cytoplasmic fraction; Nuc, nucleoplasmic fraction; Chr, chromatin fraction. d,e, Western blot (d) and bar graphs showing qPCR analysis (e) of pA usage of the SCARB2 gene evaluated in cells knocked down for XRN2 targeting 3’UTR (sh-XRN2-3’UIR) and transfected with empty vector (EV), wild-type (WT) and catalytically inactive (D235A) XRN2 (n = 3). LNCaP cells stably depleted with a shRNA targeting CDS (sh-XRN2) were used as control. Fold change of distal (d-pA) relative to the proximal pA (p-pA) in the 3’UTR was calculated by the ACq method. The representative western blot (d) shows the expression of endogenous (XRN2) and recombinant (FLAG) proteins; β-actin was used as loading control. f,g, CLIP assays performed in LNCaP cells stably depleted for XRN2 (sh-XRN2) (n = 3) (f) or transfected as in d (n = 3) (g) using the Sam68 antibody or control IgCs. The RNA associated with Sam68 was quantified by qPCR using primers located upstream of regulated and non-regulated pAs and is represented as percentage (%) of input. Inc and e-g, statistical significance was calculated by unpaired Student’s t-test (two-sided). In c, sh-XRN2/Cyt P = 0.324, sh-XRN2/Nuc P = 0.058, sh-XRN2/Chr P = 0.035, sh-Sam68/Cyt P = 0.8119, sh-Sam68/Nuc P = 0.7612, sh-Sam68/Chr p = 0.6481. In e, sh-XRN2/EV p = 3.4 ×10−3, sh-XRN2-UTR/EVP = 2.1 × 10−3, sh-XRN2-UTR/XRN2WT P = 0.4198, sh-XRN2-UTR/XRN2D235A P = 0.2456. In f, Sam68(sh-scr-downreg/sh-scr-upreg) p = 4.34 ×10−5, Sam68downreg(sh-scr/sh-XRN2) P = 1.7 × 10−3, Sam68upreg(sh-scr/sh-XRN2) P = 3 × 10−4. In g, downregulated: Sam68(sh-scr+EV/sh-XRN2-3’UTR + EV) P = 2 × 10−3, Sam68(sh-scr + EV/sh-XRN2-3’UTR + XRN2WT) P = 0.0215, Sam68(sh-scr + EV/sh-XRN2-3’UTR + XRN2D235A) P = 0.1502, Sam68(sh-XRN2-3’UTR + XRN2WT/sh-XRN2-3’UTR + EV) P = 0.0252, Sam68(sh-XRN2-3’UTR + XRN2D235A/sh-XRN2-3’UTR + EV) P = 0.0157; upregulated: Sam68(sh-scr + EV/sh-XRN2-3’UTR + EV) P = 7.3 × 10−5, Sam68(sh-scr + EV/sh-XRN2-3’UTR + XRN2WT) P = 0.036, Sam68(sh-scr + EV/sh-XRN2-3’UTR + XRN2D235A) P = 0.031, Sam68(sh-XRN2-3’UTR + XRN2WT/sh-XRN2-3’UTR + E V) p = 3.3 × 10−3, Sam68(sh-XRN2-3’UTR + XRN2D235A/sh-XRN2-3’UTR + EV) P = 0.0141. In c and e-g, the bars represent mean + s.d. of three biological replicates; statistical value is reported as *P < 0.05, **P < 0.01, ***P < 0.001; NS, not significant.

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: Modification, Western Blot, Fractionation, Stable Transfection, Transfection, Plasmid Preparation, shRNA, Expressing, Recombinant

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: RNA Binding Assay

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: a, Pearson’s correlation analysis of XRN2 and MYC expression in the jenkins dataset (GSE46691). Pearson’s correlation coefficient (r; two-sided) and P values are reported (95% confidence interval), b, Distribution of XRN2 expression in patients with PC classified as MYCflow (blue circles) and MYChigh (red squares) groups according to Z-score normalization of expression data retrieved from the jenkins dataset (GSE46691). Statistical significance was calculated by Mann-Whitney test (two-sided), and the P value is reported, c, Representative semiquantitative (sq) PCR analysis of ChIP experiments (n = 3) performed in LNCaP cells using MYC antibody and IgG, or no antibody (−), as negative controls. MYC binding was evaluated on the XRN2 promoter. Binding to the sam68 promoter and 16q22 intergenic region were used as positive and negative control, respectively. A schematic representation of the indicated promoters and 16q22 intergenic region is also shown. MYC binding sites (boxes), and positions of primers used for PCR analyses (arrows) are reported. d,e, qPCR (d) and western-blot (e) analyses of MYC, XRN2 and Sam68 expression in LNCaP and 22Rv1 cells lines transfected with control (si-scr#l) and MYC (si-MYC#1) siRNAs (n = 3). Expression was reported as fold change (ΔΔCq) with respect to control. Data represent mean + s.d. of three biological replicates, and statistical significance was calculated by unpaired Student’s t-test (two-sided) (MYC/LNCaP P = 3.8 × 10−5, MYC/22Rv1 P = 5.1 × 10−6; XRN2/LNCaP P = 3.7 × 10−3, XRN2/22Rv1 P = 1.4 × 10−3; Sam68/LNCaP P = 8.4 × 10−5, Sam68/22Rv1P = 7.7 × 10−5). In d, statistical value is reported as **P < 0.01, ***P < 0.001. In e, β-actin was used as loading control.

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: Expressing, MANN-WHITNEY, Binding Assay, Negative Control, Western Blot, Transfection

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: a, Meta-transcriptome profiles of Sam68 binding across mRNA transcripts retrieved from two replicates of CLIP-seq experiments (GSE85164). TSS, transcription start site; TES, transcription end site; RPM, reads per million, b, Representative western-blot analysesofthe co-IP ofSam68 and XRN2 with componentsoftheC/P complex from LNCaP nuclear extracts using Sam68 (α-Sam68) and XRN2 (α-XRN2) antibodies, or rabbit immunoglobulins G (α-IgG) as negative control (n = 2). c, Bar graphs representing the percentage of genes (left) and polyadenylation sites (pAs; right graph) undergoing APA regulation in Sam68 (si-Sam68)- and XRN2 (si-XRN2)-depleted LNCaP cells, d, Venn diagram showing the overlap between regulated APA events identified in Sam68- or XRN2-depleted cells. Statistical significance was calculated by hypergeometric test and the P value is shown. e, Venn diagram showing the number of unique and common up- (purple) and downregulated (orange) APA events identified in Sam68- and XRN2-depleted cells. f,g, Bar graphs showing qPCR analysis of pA usage evaluated in two representative genes undergoing 3’UTR-APA (f) and CDS-APA (g) regulation in cells knocked down for Sam68 (si-Sam68), XRN2 (si-XRN2) or both proteins. Fold change of distal (d-pA) (f) or intronic (g) pA relative to the proximal pA (p-pA) in the 3’UTR was calculated by the ΔCq method. Data represent mean + s.d. of three biological replicates. Statistical significance was calculated by unpaired Student’s t-test (two-sided). In f, SCARB2: si-Sam68/si-scr P = 1.5 × 10−3, si-XRN2/si-scr P = 2.0 × 10−3, si-Sam68si-XRN2/si-scr P = 0.017; FLNB: si-Sam68/si-scr P = 0.015, si-XRN2/si-scr P = 2.1 × 10−3, si-Sam68si-XRN2/si-scr P = 3 × 10−4. In g, RNF130: si-Sam68/si-scr P = 0.013, si-XRN2/si-scr P = 5.5 × 10−3, si-Sam68si-XRN2/si-scr P = 5.4 × 10−3; CEP70: si-Sam68/si-scr P = 4.3 × 10−3, si-XRN2/si-scr P = 0.0112, si-Sam68si-XRN2/si-scr P = 0.0147. In f and g, statistical values are reported as *P < 0.05; **P < 0.01; ***P < 0.001. UCSC genome browser tracks showing APA regulation of the events analyzed are also shown on the left side of each graph. Purple and orange boxes in the schemes indicate up- and downregulated events, respectively. Schematic representations of these CDS- and 3’UTR-APA events are shown in the upper panels.

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: Binding Assay, Western Blot, Co-Immunoprecipitation Assay, Negative Control

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: Genome-wide regulation of APA by XRN2 and Sam68 in PC cells (Related to Fig. 4).

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: Genome Wide

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: RNA Binding Assay

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: a, Percentage and number of up- (purple) and downregulated (orange) 3’UTR-APA events regulated by Sam68 and XRN2 (pA position is shown as F, proximal-most; M, intermediate; L, distal-most), b, Changes of 3’UTR pA isoform abundance (ΔAbn) at both p-pA and d-pA sites in si-Sam68 and si-XRN2 cells. Mean values and number of pA events (n) are reported, c, Percentage of up- and downregulated canonical and non-canonical PAS sequences in 3’UTR-APA events regulated by Sam68 and XRN2. d, AAUAAA frequency profile in up- (purple), down- (orange) and unregulated (black) 3’UTR pAs evaluated between −100 and +100 nt from the CS (shading represents 95% confidence interval). Statistical significance (unpaired Student’s t-test, two-sided) was calculated between −15 and −25 nt (boxplot). e, A- and G-base frequency distribution in up- (purple), down-grange) and unregulated (black) pAs between −100 and +100 nt from the CS (0). f, Scheme of cis-elements and CS position. Hexamers enriched between −100 and +100 nt from the CS in up- and downregulated pAs with respect to unregulated pAs. Motif (H), number (N) and significance score (P) of hexamers are indicated. Significance score was calculated by –log10(P)xS, where P is based on the Fisher’s exact test and the S value was 1 or −1 for enrichment and depletion, respectively, g, APA isoform abundance (Abn) of si-Sam68/si-XRN2 up- (mean = 28.6) and downregulated (mean = 47.2) isoforms. Values refer to expression in control cells, h, Scheme of the FLNB minigene comprising the genomic region from the second-last exon to 200 nt downstream of the d-pA (source data). i,j, Semiquantitative (micrographs) and quantitative (bar graphs) analyses of pA usage in LNCaP transfected with the FLNB minigene and indicated plasmids (n = 3). Protein expression was evaluated by western blot, k, CLIP assays performed in sh-Sam68 and sh-XRN2 cells using CPSF30 antibody or IgGs (n = 3). Statistical significance was calculated by unpaired Student’s t-test, two-sided (b, g, i-k) and with Fisher’s exact test, two-sided (a, c). (l-k) Bar graphs represent mean + s.d. When not indicated, P values are reported as *P < 0.05, ***P < 0.001, ****P < 0.0001 (exact P values are reported in the source data). In the boxplots (b, d, g), the center line and box indicate the median and the 25th and 75th percentiles, respectively. Whiskers indicate ±1.5x interquartile range.

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: Expressing, Transfection, Western Blot

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: a, Enrichment of Gene Ontology (GO) terms (dot plot) in genes regulated by 3’UTR-APA upon depletion of Sam68 or XRN2. Dot size and color indicate the number of genes and statistical significance (Fisher’s exact test, two-sided), respectively, b, Cytometric analyses showing DNA content versus BrdU incorporation upon stable depletion of Sam68 (sh-Sam68) and XRN2 (sh-XRN2) in LNCaP cells. The bar graph shows the percentage of BrdU-positive (S phase) cells, c. Percentage (mean + s.d.) of BrdU-positive LNCaP cells described in b at the indicated time points after release from G1/S synchronization. d,e, Western blot (d) and qPCR (e) analyses of MCM10 and ORC2 expression level in sh-Sam68 and sh-XRN2 LNCaP cells (n = 3). f, PCR strategy used to evaluate 3’UTR-APA isoforms distribution on a 15–50% sucrose gradient, g, sqPCR analysis of the indicated p-pA and d-pA isoform abundance within the polysomal and non-polysomal fractions obtained from sucrose gradient. The graphs show the densitometric analysis of the band signal in each fraction, expressed as a percentage of that detected in all fractions, h, Relative luciferase activity (Renilla/Firefly ratio) of long and short MCM10 3’UTR in LNCaP cells. i, Representative western-blot analysis (n = 3) of the indicated proteins performed in LNCaP cells depleted for the indicated genes, j, Cytometric analyses showing DNA content versus BrdU incorporation in control (si-scr), si-MCMlO and si-ORC2 LNCaP cells. The bar graph shows the percentage of S-phase BrdU-positive cells, k, Kaplan-Meier curves comparing progression-free survival of494 patients with PC (Prostate Adenocarcinoma, TCGA, PanCancer Atlas; https://www.cbioportal.org) stratified according to MCM10 (right), ORC2 (middle) and MCM10/ORC2 (left) expression level. I, Schematic model showing the impact of the functional interaction between Sam68 and XRN2 on cell cycle regulation. The Sam68/XRN2 complex promotes 3’UTR shortening of cell cycle-related genes, increasing their mRNA translation efficiency and cell proliferation. Conversely, Sam68/XRN2 knockdown induces 3’UTR lengthening, reduces translation efficiency of transcripts and causes cell cycle arrest. In b, e, h and j, the bar graphs represent the mean + s.d. In b, c, e, g, h and j, statistical significance was calculated by unpaired Student’sf-test, two-sided (n = 3; *P < 0.05, **P < 0.01,***P < 0.001; NS, not significant; exactPvalues are reported in the source data). In d and I, β-actin was used as loading control.

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: BrdU Incorporation Assay, Western Blot, Expressing, Luciferase, Activity Assay, Functional Assay

Journal: Nature structural & molecular biology

Article Title: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer

doi: 10.1038/s41594-022-00853-0

Figure Lengend Snippet: From: The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polvadenvlation to cell cycle progression in prostate cancer

Article Snippet: Immunostaining for Sam68 and XRN2 Five-micrometer sections from formalin-fixed and paraffin-embedded human PC samples ( n = 20) were deparaffinized, rehydrated and stained with rabbit polyclonal antibodies raised against human Sam68 (1:2,000 dilution, overnight incubation; cat. no. A302-110A, Bethyl Laboratories) and human XRN2 (1:400 dilution, overnight incubation; cat. no. A301-103A, Bethyl Laboratories).

Techniques: RNA Binding Assay

Journal: Theranostics

Article Title: SAM68 promotes tumorigenesis in lung adenocarcinoma by regulating metabolic conversion via PKM alternative splicing

doi: 10.7150/thno.51360

Figure Lengend Snippet: SAM68 is up-regulated in Lung adenocarcinoma (LUAD) and correlates with a poor prognosis for LUAD patients. (A~C) SAM68 mRNA levels was up-regulated in LUAD compared to normal lung tissue based on the Oncomine, GEO and TCGA database. (D~G) From TCGA LUAD specimen cohorts, compared with the patients with low expression level of SAM68 , the patients with high mRNA expression of SAM68 had higher recurrence rates and death rates, shorter RFS, and OS. (H~J) The SAM68 mRNA and protein levels were detected by qPCR, RT-PCR and Western blot in the LUAD tissues (T) and their corresponding adjacent non-tumoral tissues (N). (K) IHC analysis of the expression of SAM68 protein was represented in LUAD tissues and the corresponding adjacent normal lung tissues. (L) Differences in expression levels of SAM68 protein in LUAD tissues (n = 50) and adjacent normal lung tissues (n = 50). Two-tailed t-tests were used B, C, H and L. Pearson Chi-Square tests were used in D and F.Log rank tests were used in E and G.

Article Snippet: In short, after dewaxing, the tissue microarray chip was treated with 3% hydrogen peroxide in methanol and blocked with a standard labeled streptavidin biotin kit (DAKO, Germany), incubated overnight with SAM68 polyclonal antibody (proteintech, 10222-1-AP, 1:100) in a moist chamber at 4 °C.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Two Tailed Test

Journal: Theranostics

Article Title: SAM68 promotes tumorigenesis in lung adenocarcinoma by regulating metabolic conversion via PKM alternative splicing

doi: 10.7150/thno.51360

Figure Lengend Snippet: Downregulation of SAM68 inhibited the malignant phenotypes of LUAD cells in vitro and tumorigenesis and progression in vivo . (A and B) NCI-H1975 and A549 cells were transfected with anti-SAM68 siRNAs, SAM68 mRNA (A) and protein levels (B) level were determined by qPCR and Western blot, respectively. (C) After SAM68 silencing, SAM68 was immuno-stained with anti-SAM68 antibody in NCI-H1975 cells. (D-F) The effects of SAM68 silencing on NCI-H1975 and A549 cells growth (D), colony formation (E), and migration and invasion (F) were detected. (G) CRISPR-Cas9 mediated knockout (KO) of SAM68 in NCI-H1975 cells as detected by immunofluorescence. (H) The in vivo growth of SAM68 KO (sgSAM68) NCI-H1975 cellswas detected (n = 6). (I)The in vivo lung metastasis of SAM68 KO NCI-H1975 cells was examined (n = 5). (J and K) The hematoxylin and eosin (HE) staining and Kaplan-Meier curves are shown for two cohorts of transplanted mice carrying SAM68 KO cells and control groups. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01 or ***p < 0.001. Two-tailed t-tests were used A, E, F and I. Two-way ANOVA was used in D. Log rank tests was used in L.

Article Snippet: In short, after dewaxing, the tissue microarray chip was treated with 3% hydrogen peroxide in methanol and blocked with a standard labeled streptavidin biotin kit (DAKO, Germany), incubated overnight with SAM68 polyclonal antibody (proteintech, 10222-1-AP, 1:100) in a moist chamber at 4 °C.

Techniques: In Vitro, In Vivo, Transfection, Western Blot, Staining, Migration, CRISPR, Knock-Out, Immunofluorescence, Control, Two Tailed Test

Journal: Theranostics

Article Title: SAM68 promotes tumorigenesis in lung adenocarcinoma by regulating metabolic conversion via PKM alternative splicing

doi: 10.7150/thno.51360

Figure Lengend Snippet: The 351~443 motif of SAM68 binds to the Glycine residues in RGG box of hnRNP A1. (A) HEK293T cells were transfected with Flag-SAM68. After Co-IP, proteins that interacted with Flag-SAM68 were identified using silver staining combined mass spectrometry. (B) The SAM68-interacting proteins were performed by GO analysis. (C and D) The unique peptide of SAM68 (C) and hnRNP (D) A1 were identified by mass spectrometry. (E and F) Flag-SAM68 and HA-hnRNP A1 plasmid were transfected into NCI-H1975 cells, cellular lysates were treated with 10 mg/mL RNase A (Thermofisher, EN0531) for 1 h or no treatment, Flag-SAM68 complexes were co-immunoprecipitated by anti-Flag antibody, then hnRNP A1 was detected (E), and HA-hnRNP A1 complexes were co-immunoprecipitated by anti-HA antibody, then SAM68 was detected (F). (G) Confocal images of endogenous SAM68 and hnRNP A1 in NCI-H1975 cells. (H) Wild-type (WT) and indicated mutations with the different domain of hnRNP A1 were constructed. (I and J) The indicated HA-hnRNP A1 WT and mutation constructs and Flag-SAM68 were co-transfected into HEK293T cells, Flag-SAM68 and HA-hnRNP A1 complexes were co-immunoprecipitated by anti-Flag and HA antibody, respectively; HA-hnRNP A1 mutants were detected using anti-HA antibodies, and Flag-SAM68 were detected using anti-Flag antibodies. (K and L) The HA-hnRNP A1 WT or its RAA mutation with Flag-SAM68 were co-transfected into HEK293T cells, and the interactions of hnRNP A1 RAA mutant with Flag-SAM68 were detected as described in (I) and (J). (M and N) The Flag-SAM68 WT or its indicated mutation with HA-hnRNP A1 were co-transfected into HEK293T cells, the interactions of Flag-SAM68 indicated mutation with HA-hnRNP A1 were detected as described in (I) and (J).

Article Snippet: In short, after dewaxing, the tissue microarray chip was treated with 3% hydrogen peroxide in methanol and blocked with a standard labeled streptavidin biotin kit (DAKO, Germany), incubated overnight with SAM68 polyclonal antibody (proteintech, 10222-1-AP, 1:100) in a moist chamber at 4 °C.

Techniques: Transfection, Co-Immunoprecipitation Assay, Silver Staining, Mass Spectrometry, Plasmid Preparation, Immunoprecipitation, Construct, Mutagenesis

Journal: Theranostics

Article Title: SAM68 promotes tumorigenesis in lung adenocarcinoma by regulating metabolic conversion via PKM alternative splicing

doi: 10.7150/thno.51360

Figure Lengend Snippet: Silencing hnRNP A1 antagonized the enhancement of malignant phenotypes induced by SAM68 overexpression. (A~D) The anti-hnRNP A1 siRNAs were transfected into NCI-H1975 and A549 cells, hnRNP A1 protein levels (A) level was detected by Western blot, and the effects of silencing hnRNP A1 on NCI-H1975 and A549 cells growth (B), colony formation (C), migration and invasion (D) were detected. (E~H) The Flag-SAM68 plasmid and anti-hnRNPA1 siRNAs were co-transfected into NCI-H1975 and A549 cells, the indicated protein levels (E), cell growth (F), colony formation (G), migration and invasion (H) were detected. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01 or ***p < 0.001. Two-way ANOVA were used in B and F, two-tailed t-tests were used C, D, G and H.

Article Snippet: In short, after dewaxing, the tissue microarray chip was treated with 3% hydrogen peroxide in methanol and blocked with a standard labeled streptavidin biotin kit (DAKO, Germany), incubated overnight with SAM68 polyclonal antibody (proteintech, 10222-1-AP, 1:100) in a moist chamber at 4 °C.

Techniques: Over Expression, Transfection, Western Blot, Migration, Plasmid Preparation, Two Tailed Test

Journal: Theranostics

Article Title: SAM68 promotes tumorigenesis in lung adenocarcinoma by regulating metabolic conversion via PKM alternative splicing

doi: 10.7150/thno.51360

Figure Lengend Snippet: The 351~443 motif of SAM68 favors the binding of the RGG motif of hnRNP A1 to the intronic sequences flanking exon 9 (EI9) by binding to the Glycine residues in RGG box of hnRNP A1. (A) Quantification of SAM68-regulated AS events in each category was measured RNA sequencing. (A3SS/A5SS, alternative 3'/5' splice sites, MXE, mutually exclusive exons, RI, retained introns, SE, skipped exons) (B) Changes in PSI values of SAM68-regulated AS events were shown. (C) SAM68 regulated PKM pre-mRNA splicing and promoted PKM2 isoform formation. (D) CLIP assay of SAM68 and hnRNP A1 binding to the PKM pre-mRNA. NCI-H1975 cells were UV-crosslinked and immunoprecipitated with control IgGs or antibodies, as indicated. (E) RNA affinity purification followed by Western blot showed in vitro binding of the indicated biotin-labeled RNAs with endogenous hnRNP A1 or SAM68. (F) The Flag-SAM68 plasmid at the indicated doses was transfected into NCI-H1975 cells, and RNA affinity purification was performed using biotin-labeled RNA EI9 (50-68). (G) NCI-H1975 cells were co-transfected with Flag-SAM68 plasmid and the indicated hnRNP A1 mutations, and in vitro binding of EI9 (50-68) RNA probes with WT or the indicated hnRNP A1 mutations was detected. (H) The HA-hnRNP A1 WT or its RAA mutation was transfected into NCI-H1975 cells, and RNA affinity purification was performed using biotin-labeled RNA EI9 (50-68). (I) HA-hnRNP A1 WT or its RAA mutation with Flag-SAM68 plasmids were co-transfected into NCI-H1975 cells, and in vitro binding of EI9 (50-68) RNA probes with WT or its RAA mutation was detected. (J) The WT SAM68 or its indicated mutant with WT hnRNP A1-HA plasmids were cotransfected into NCI-H1975 cells, and RNA affinity purification was performed using biotin-labeled RNA EI9 (50-68).

Article Snippet: In short, after dewaxing, the tissue microarray chip was treated with 3% hydrogen peroxide in methanol and blocked with a standard labeled streptavidin biotin kit (DAKO, Germany), incubated overnight with SAM68 polyclonal antibody (proteintech, 10222-1-AP, 1:100) in a moist chamber at 4 °C.

Techniques: Binding Assay, RNA Sequencing, Immunoprecipitation, Control, Affinity Purification, Western Blot, In Vitro, Labeling, Plasmid Preparation, Transfection, Mutagenesis

Journal: Theranostics

Article Title: SAM68 promotes tumorigenesis in lung adenocarcinoma by regulating metabolic conversion via PKM alternative splicing

doi: 10.7150/thno.51360

Figure Lengend Snippet: Sam68 and hnRNP A1 cooperated in PKM splicing to decrease PKM1 isoform formation and increase PKM2 isoform formation, then promoting the aerobic glycolysis of LUAD cells. (A and B) NCI-H1975 cells were transfected with the Flag-SAM68 plasmids (A) or anti-SAM68 siRNAs (B), followed by the PKM splicing assay. (C) PKM splicing was performed in SAM68 KO NCI-H1975 cells. (D) The Flag-SAM68 plasmid and anti-hnRNPA1 siRNAs were co-transfected into NCI-H1975, followed by the PKM splicing assay. (E) PKM splicing was performed in the LUAD tissue samples with low (n = 6) and high (n = 6) SAM68 expression. (F) The ratio of PKM2/PKM1 is calculated in the LUAD tissue samples with low (n = 6) and high (n = 6) SAM68 expression (G and H) NCI-H1975 cells were transfected with the Flag-SAM68 plasmids, glucose uptake and lactate production were measured. (I and J) Glucose uptake and lactate production were measured in SAM68 KO NCI-H1975 cells. (K and L) NCI-H1975 cells were transfected with anti- hnRNP A1 siRNAs, then glucose uptake (K) and lactate production (L) were detected. (M and N) The Flag-SAM68 plasmid and anti-hnRNPA1 siRNAs were co-transfected into NCI-H1975, and glucose uptake (M) and lactate production (N) were detected. (O and P) The Flag-SAM68 plasmid and anti-PKM2 siRNAs were co-transfected into NCI-H1975, and glucose uptake (O) and lactate production (P) were detected.

Article Snippet: In short, after dewaxing, the tissue microarray chip was treated with 3% hydrogen peroxide in methanol and blocked with a standard labeled streptavidin biotin kit (DAKO, Germany), incubated overnight with SAM68 polyclonal antibody (proteintech, 10222-1-AP, 1:100) in a moist chamber at 4 °C.

Techniques: Transfection, Splicing Assay, Plasmid Preparation, Expressing

Journal: Theranostics

Article Title: SAM68 promotes tumorigenesis in lung adenocarcinoma by regulating metabolic conversion via PKM alternative splicing

doi: 10.7150/thno.51360

Figure Lengend Snippet: SAM68 increases PKM2 isoform formation, and promotes malignant phenotypes and aerobic glycolysis by cooperating to hnRNP A1 . The WT SAM68 or its indicated mutant was transfected into SAM68 KO NCI-H1975 cells; the indicated proteins were detected by Western blot (A), and cell growth (B), colony formation (C and D), migration and invasion (E), PKM splicing (F), glucose uptake (G), and lactate production (H) were detected. (I) A regulatory model of SAM68 on tumorigenesis proposed in this study. Data are represented as mean ± SEM. **p < 0.01 or ***p < 0.001. Two-way ANOVA was used in B; two-tailed t-tests were used in D~ H.

Article Snippet: In short, after dewaxing, the tissue microarray chip was treated with 3% hydrogen peroxide in methanol and blocked with a standard labeled streptavidin biotin kit (DAKO, Germany), incubated overnight with SAM68 polyclonal antibody (proteintech, 10222-1-AP, 1:100) in a moist chamber at 4 °C.

Techniques: Mutagenesis, Transfection, Western Blot, Migration, Two Tailed Test

Journal:

Article Title: Sam68 Enhances the Cytoplasmic Utilization of Intron-Containing RNA and Is Functionally Regulated by the Nuclear Kinase Sik/BRK

doi: 10.1128/MCB.23.1.92-103.2003

Figure Lengend Snippet: Sam68 enhances CTE function in 293 and COS cells. Five micrograms of the pCMVGag-Pol-CTE reporter plasmid (pHR1361) and 0.25 μg of pCMVSEAP (pHR1831) were transfected into 100-mm-diameter dishes of either 293 cells (A) or COS cells (B) together with 1 μg each of pcDFSam68 (pHR2208), pcDHSam68ΔKH (pHR2212), pcDHSam68(G178E) (pHR2210), or pcDNA3 with no insert (pHR2214). Supernatants were collected at 72 h posttransfection, and p24 and SEAP levels in each sample were determined. p24 values were then adjusted for variations in SEAP levels. The data represent the average of two independent transfections.

Article Snippet: Sam68 was immunoprecipitated from 200 μg of total cell lysates with 1 μg of mouse anti-Sam68 monoclonal antibody (SC-333; Santa Cruz) and 30 μl of 50% protein G-Sepharose beads (Amersham Biosciences) after incubation at 4°C for 3 h. Samples were washed two times with 500 μl of lysis buffer and one time with 500 μl of 10 mM Tris HCl-100 mM NaCl-1 mM EDTA (pH 8.0) (TNE) with centrifugation at 500 × g for 3 min, resuspended in 1.5× sodium dodecyl sulfate (SDS) sample buffer, boiled for 5 min, centrifuged briefly, and resolved by SDS-polyacrylamide gel electrophoresis (PAGE) on a 12% gel.

Techniques: Plasmid Preparation, Transfection

Journal:

Article Title: Sam68 Enhances the Cytoplasmic Utilization of Intron-Containing RNA and Is Functionally Regulated by the Nuclear Kinase Sik/BRK

doi: 10.1128/MCB.23.1.92-103.2003

Figure Lengend Snippet: Comparison of endogenous Sam68 produced in 293T and COS cells. Sam68 was immunoprecipitated with anti-Sam68 monoclonal antibodies from lysates of 293T and COS cells metabolically labeled with [35S]Met/Cys Tran35S-label for either 3 or 24 h. Protein was resolved by SDS-PAGE and visualized by PhosphorImager analysis. IgG1, immunoglobulin G1.

Article Snippet: Sam68 was immunoprecipitated from 200 μg of total cell lysates with 1 μg of mouse anti-Sam68 monoclonal antibody (SC-333; Santa Cruz) and 30 μl of 50% protein G-Sepharose beads (Amersham Biosciences) after incubation at 4°C for 3 h. Samples were washed two times with 500 μl of lysis buffer and one time with 500 μl of 10 mM Tris HCl-100 mM NaCl-1 mM EDTA (pH 8.0) (TNE) with centrifugation at 500 × g for 3 min, resuspended in 1.5× sodium dodecyl sulfate (SDS) sample buffer, boiled for 5 min, centrifuged briefly, and resolved by SDS-polyacrylamide gel electrophoresis (PAGE) on a 12% gel.

Techniques: Produced, Immunoprecipitation, Metabolic Labelling, Labeling, SDS Page

Journal:

Article Title: Sam68 Enhances the Cytoplasmic Utilization of Intron-Containing RNA and Is Functionally Regulated by the Nuclear Kinase Sik/BRK

doi: 10.1128/MCB.23.1.92-103.2003

Figure Lengend Snippet: Sam68 enhances CTE function in a stable 293T packaging cell line and Qcl3 quail cells. (A) Dishes (100-mm diameter) of B2.10 cells were transfected with 1 μg of pcDFSam68 (pHR2208) or pcDNA empty vector (pHR2214). B2.10 is a cell line derived from 293T cells by stable transfection of pCMVGag-Pol-CTE (pHR1361) and pCMVEnv-CTE (pHR1374). p24 levels in the supernatant were determined at 72 h posttransfection. The data represent the average of two independent transfections. (B) Dishes (100-mm diameter) of Qcl3 quail fibroblast cells were transfected with 5 μg of the pCMVGag-Pol-CTE reporter (pHR1361) and 0.25 μg of pCMVSEAP (pHR1831) together with 2 μg of pCMVTap (pHR2128), 1 μg of pcDFSam68 (pHR2208), or both. At 72 h posttransfection, supernatants were collected and analyzed for p24 and SEAP expression. The data represent the average of two independent transfections.

Article Snippet: Sam68 was immunoprecipitated from 200 μg of total cell lysates with 1 μg of mouse anti-Sam68 monoclonal antibody (SC-333; Santa Cruz) and 30 μl of 50% protein G-Sepharose beads (Amersham Biosciences) after incubation at 4°C for 3 h. Samples were washed two times with 500 μl of lysis buffer and one time with 500 μl of 10 mM Tris HCl-100 mM NaCl-1 mM EDTA (pH 8.0) (TNE) with centrifugation at 500 × g for 3 min, resuspended in 1.5× sodium dodecyl sulfate (SDS) sample buffer, boiled for 5 min, centrifuged briefly, and resolved by SDS-polyacrylamide gel electrophoresis (PAGE) on a 12% gel.

Techniques: Transfection, Plasmid Preparation, Derivative Assay, Stable Transfection, Expressing

Journal:

Article Title: Sam68 Enhances the Cytoplasmic Utilization of Intron-Containing RNA and Is Functionally Regulated by the Nuclear Kinase Sik/BRK

doi: 10.1128/MCB.23.1.92-103.2003

Figure Lengend Snippet: Coexpression of the constitutively active Sik(Y447F) mutant protein with Sam68 inhibits Sam68-mediated enhancement of CTE function. (A) Five micrograms of pCMVGag-Pol-CTE reporter (pHR1361) and 0.25 μg of pCMVSEAP (pHR1831) were transfected into 293T cells with 1 μg of a plasmid that expresses either the wild-type (pcDNA3-Sikwt, pHR2531), the kinase-inactive [pcDNA3-Sik(K219M), pHR2533], or the constitutively active [pcDNA3Sik(Y447F), pHR2533] form of Sik. These plasmids were expressed either alone or together with 1 μg of pcDSam68 (pHR2208). At 72 h posttransfection, supernatants were collected and analyzed for p24 and SEAP expression. The data represent the average of two independent transfections. (B) Increasing amounts of pcDFSam68 or pcDNA3-Sik(Y447F) were cotransfected with 5 μg of pCMVGag-Pol-CTE reporter (pHR1361) and 0.25 μg of pCMVSEAP (pHR1831) plasmid into 293T cells. At 72 h posttransfection, supernatants were collected and analyzed for p24 and SEAP expression. The data represent the average of two independent transfections.

Article Snippet: Sam68 was immunoprecipitated from 200 μg of total cell lysates with 1 μg of mouse anti-Sam68 monoclonal antibody (SC-333; Santa Cruz) and 30 μl of 50% protein G-Sepharose beads (Amersham Biosciences) after incubation at 4°C for 3 h. Samples were washed two times with 500 μl of lysis buffer and one time with 500 μl of 10 mM Tris HCl-100 mM NaCl-1 mM EDTA (pH 8.0) (TNE) with centrifugation at 500 × g for 3 min, resuspended in 1.5× sodium dodecyl sulfate (SDS) sample buffer, boiled for 5 min, centrifuged briefly, and resolved by SDS-polyacrylamide gel electrophoresis (PAGE) on a 12% gel.

Techniques: Mutagenesis, Transfection, Plasmid Preparation, Expressing

Journal:

Article Title: Sam68 Enhances the Cytoplasmic Utilization of Intron-Containing RNA and Is Functionally Regulated by the Nuclear Kinase Sik/BRK

doi: 10.1128/MCB.23.1.92-103.2003

Figure Lengend Snippet: Expression of the Sik(Y447F) mutant increases phosphorylation of Sam68 at multiple residues. 293T cells were transfected with plasmids expressing Flag-Sam68 either alone or with a plasmid expressing Sik(Y447F) and labeled with [32P]orthophosphate. (A, left side) 32P analysis of immunoprecipitated Sam68. Sam68 was immunoprecipitated from transfected cells by using an agarose bead-conjugated anti-FLAG antibody. Proteins were resolved by SDS-PAGE, and 32P incorporation was visualized by PhosphorImager analysis. (A, right side) Western blot analysis of Sam68 in the absence or presence of Sik(Y447F). Proteins from whole-cell lysates were resolved by SDS-PAGE and transferred to Immobilon. The blot was probed with an anti-Sam68 monoclonal antibody, and Sam68 was visualized by using an alkaline phosphatase-conjugated anti-mouse secondary antibody. (B) Two-dimensional phosphopeptide analysis of Sam68 expressed in the presence or absence of Sik(Y447F). Tryptic peptide analysis was carried out on Sam68 immunoprecipitated from 32P-labeled 293T cells by using an agarose bead-conjugated anti-FLAG antibody. The peptides were resolved in two dimensions, first by electrophoresis in pH 1.9 buffer and second by chromatography in phosphochromatography buffer. Labeled peptides were visualized by PhosphorImager analysis. Arrows indicate peptides that appeared de novo or increased in intensity in the presence of Sik(Y447F).

Article Snippet: Sam68 was immunoprecipitated from 200 μg of total cell lysates with 1 μg of mouse anti-Sam68 monoclonal antibody (SC-333; Santa Cruz) and 30 μl of 50% protein G-Sepharose beads (Amersham Biosciences) after incubation at 4°C for 3 h. Samples were washed two times with 500 μl of lysis buffer and one time with 500 μl of 10 mM Tris HCl-100 mM NaCl-1 mM EDTA (pH 8.0) (TNE) with centrifugation at 500 × g for 3 min, resuspended in 1.5× sodium dodecyl sulfate (SDS) sample buffer, boiled for 5 min, centrifuged briefly, and resolved by SDS-polyacrylamide gel electrophoresis (PAGE) on a 12% gel.

Techniques: Expressing, Mutagenesis, Transfection, Plasmid Preparation, Labeling, Immunoprecipitation, SDS Page, Western Blot, Electrophoresis, Chromatography

Journal:

Article Title: Sam68 Enhances the Cytoplasmic Utilization of Intron-Containing RNA and Is Functionally Regulated by the Nuclear Kinase Sik/BRK

doi: 10.1128/MCB.23.1.92-103.2003

Figure Lengend Snippet: Analysis of p24 and Gag-Pol RNA in 293T cells. 293T cells were transfected with the reporter plasmids pCMVGag-Pol-RRE (pHR354) or pCMVGag-Pol-CTE (pHR1361) and pCMVSEAP (pHR1831) in the presence or absence of Rev (pHR30)- and/or Sam68 (pHR2208)-expressing plasmids essentially as described in the legend to Fig. ​Fig.1.1. (A) Supernatants were harvested at 72 h posttransfection, and p24 levels were analyzed by ELISA. (B) Cells were harvested at 72 h and analyzed by Western blot assay. The Western blot analysis was performed on whole-cell lysates prepared from transfected cells with an anti-p24 primary monoclonal antibody, a rabbit anti-mouse secondary antibody, and 125I-coupled protein A for detection. Brackets show the fold difference in p24 levels between indicated lanes. (C) Northern blot analysis of cytoplasmic and total poly(A)+ RNA isolated from 293T cells 60 h after transfection. Gag-Pol- and SEAP-specific mRNAs were detected by using specific radiolabeled DNA probes. The blot was analyzed with a Molecular Dynamics PhosphorImager and ImageQuant software. Brackets show the fold differences in the levels of the Gag-Pol RNA bands between the indicated lanes after adjustment for variations in SEAP RNA levels.

Article Snippet: Sam68 was immunoprecipitated from 200 μg of total cell lysates with 1 μg of mouse anti-Sam68 monoclonal antibody (SC-333; Santa Cruz) and 30 μl of 50% protein G-Sepharose beads (Amersham Biosciences) after incubation at 4°C for 3 h. Samples were washed two times with 500 μl of lysis buffer and one time with 500 μl of 10 mM Tris HCl-100 mM NaCl-1 mM EDTA (pH 8.0) (TNE) with centrifugation at 500 × g for 3 min, resuspended in 1.5× sodium dodecyl sulfate (SDS) sample buffer, boiled for 5 min, centrifuged briefly, and resolved by SDS-polyacrylamide gel electrophoresis (PAGE) on a 12% gel.

Techniques: Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Northern Blot, Isolation, Software

Journal:

Article Title: Sam68 Enhances the Cytoplasmic Utilization of Intron-Containing RNA and Is Functionally Regulated by the Nuclear Kinase Sik/BRK

doi: 10.1128/MCB.23.1.92-103.2003

Figure Lengend Snippet: In situ hybridization of Gag-Pol-CTE RNA in cells with or without Sam68 coexpression. Cells were transfected with plasmids expressing Gag-Pol-RRE RNA (with or without Rev) or Gag-Pol-CTE RNA (with or without Sam68), as indicated. At 72 h after transfection, cells were fixed and hybridized to an antisense DIG-labeled oligonucleotide probe specific for a sequence within gag. Hybridization was detected with an anti-DIG antibody conjugated to FITC and visualized by LSM5 Pascal confocal microscopy (Zeiss).

Article Snippet: Sam68 was immunoprecipitated from 200 μg of total cell lysates with 1 μg of mouse anti-Sam68 monoclonal antibody (SC-333; Santa Cruz) and 30 μl of 50% protein G-Sepharose beads (Amersham Biosciences) after incubation at 4°C for 3 h. Samples were washed two times with 500 μl of lysis buffer and one time with 500 μl of 10 mM Tris HCl-100 mM NaCl-1 mM EDTA (pH 8.0) (TNE) with centrifugation at 500 × g for 3 min, resuspended in 1.5× sodium dodecyl sulfate (SDS) sample buffer, boiled for 5 min, centrifuged briefly, and resolved by SDS-polyacrylamide gel electrophoresis (PAGE) on a 12% gel.

Techniques: In Situ Hybridization, Transfection, Expressing, Labeling, Sequencing, Hybridization, Confocal Microscopy

Journal:

Article Title: Sam68 Enhances the Cytoplasmic Utilization of Intron-Containing RNA and Is Functionally Regulated by the Nuclear Kinase Sik/BRK

doi: 10.1128/MCB.23.1.92-103.2003

Figure Lengend Snippet: Effect of Sam68 on Rev/RRE function. (A) Five micrograms of pCMVGag-Pol-RRE (pHR354) and 0.25 μg of pCMVSEAP (pHR1831) were transfected into 100-mm-diameter dishes of 293 cells with the indicated amounts of pCMVRev (pHR30) with or without 1 μg of pcDFSam68 (pHR2208). p24 analysis was carried out as described in the legend to Fig. ​Fig.1.1. (B) Dishes (150-mm diameter) of 293T cells were transfected with 20 μg of pCMVGag-Pol-RRE and 5 μg of pCMVSEAP in the presence or absence of 5 μg of pCMVRev and/or pcDFSam68. Northern blot analysis was performed on cytoplasmic (Cyto) and total poly(A)+ mRNA isolated at 48 h after transfection and detected with a radiolabeled gag/pol-specific probe. Gag-Pol- and SEAP-specific mRNAs were detected by using specific radiolabeled DNA probes. The blot was analyzed with a Molecular Dynamics Phosphorimager and ImageQuant software. Brackets show the fold differences in the levels of the Gag-Pol RNA bands between the indicated lanes after adjustment for variations in SEAP RNA levels.

Article Snippet: Sam68 was immunoprecipitated from 200 μg of total cell lysates with 1 μg of mouse anti-Sam68 monoclonal antibody (SC-333; Santa Cruz) and 30 μl of 50% protein G-Sepharose beads (Amersham Biosciences) after incubation at 4°C for 3 h. Samples were washed two times with 500 μl of lysis buffer and one time with 500 μl of 10 mM Tris HCl-100 mM NaCl-1 mM EDTA (pH 8.0) (TNE) with centrifugation at 500 × g for 3 min, resuspended in 1.5× sodium dodecyl sulfate (SDS) sample buffer, boiled for 5 min, centrifuged briefly, and resolved by SDS-polyacrylamide gel electrophoresis (PAGE) on a 12% gel.

Techniques: Transfection, Northern Blot, Isolation, Software

Journal: Human Molecular Genetics

Article Title: Arginine methylation of RNA-binding proteins is impaired in Huntington’s disease

doi: 10.1093/hmg/ddad125

Figure Lengend Snippet: Arginine methylation of HNRNPUL2, KHDRBS1(SAM68) and SRSF1 is reduced in HD ISPNs ( A ) Total cell lysates from non-differentiated control (33CAG) and HD (180CAG) ISPNs were prepared as described in the Experimental Section. IPs with a mixture of mono-methyl (MMA) and asymmetric dimethyl (ADMA) antibodies were performed followed by western blot analysis with indicated protein-specific antibodies. Protein bands were visualized and quantified using the Licor System and Image Studio software. ( B ) – ( D ) Graphs (left) show the mean intensity values (±SEM) of the signal detected with antibodies to total HNRNPUL2, KHDRBS1(SAM68) and SRSF1 in the IPs normalized to the signal obtained from input lysates with the same antibodies. Total protein levels normalized to β-tubulin were also quantified (right graphs). n = 3 (biological replicates), Normality Test (Shapiro–Wilk): Passed, Equal Variance Test: Passed. T -test with equal variances was performed: (B) * 33CAG versus 180CAG, P 0.04. (C) * 33CAG versus 180CAG, P 0.0046. * * 33CAG versus 180CAG, P 0.0039.

Article Snippet: FLAG-tagged HNRNPUL2 and KHDRBS1 expression plasmids were obtained from GenScript.

Techniques: Methylation, Control, Western Blot, Software

Journal: Human Molecular Genetics

Article Title: Arginine methylation of RNA-binding proteins is impaired in Huntington’s disease

doi: 10.1093/hmg/ddad125

Figure Lengend Snippet: RNA association of hypomethylated RBPs is decreased in HD cells. RNA-binding assay in ISPNs. RNA pull-downs were performed as described in the Experimental Section according to Castello et al . (2013) . ( A ) Validation of RNA-binding assay in ISPNs demonstrating detection of indicated RNA-bound proteins in cross-linked cells (but not in the eluates of nonirradiated control cells) with protein-specific antibodies. ( B ) The amounts of mRNA within RNA-protein complexes were estimated by absorbance at A260, and equal amounts were analyzed by western blotting with indicated protein-specific antibodies. Representative experiment is shown. ( C ) – ( G ) Quantitation of blots shown in (B). Graphs show the mean intensity values (±SD) of the signal with antibodies to indicated proteins detected in the RNA-bound fraction normalized to the signal obtained from input lysates with the same antibodies. n = 3 (biological replicates). Normality test (Shapiro–Wilk): Passed, equal variance test: Passed. One-way ANOVA was performed with pairwise multiple comparison procedures (Holm–Sidak method). (C) HNRNPUL2, * 33EE versus 180, P 0.002; * * 33DG versus 180, P 0.002. (D) KHDRBS1, * 33EE versus 180, P 0.004; * * 33DG versus 180, P < 0.001; * * * 33DG versus 33EE, P < 0.001. (E) FUS, * 33EE versus 180, P 0.003; * * 33DG versus 180, P 0.008. (F) SRSF1, * 33EE versus 180, P 0.003; * * 33DG versus 180, P 0.011. (G) TAF15, * 33EE versus 180, P < 0.001; * * 33DG versus 180, P < 0.001; * * * 33DG versus 33EE, P < 0.001.

Article Snippet: FLAG-tagged HNRNPUL2 and KHDRBS1 expression plasmids were obtained from GenScript.

Techniques: RNA Binding Assay, Biomarker Discovery, Control, Western Blot, Quantitation Assay, Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: RNA binding protein Sam68 promotes germinal center reaction and IgG response through regulation of miR29

doi: 10.1007/s00018-026-06145-w

Figure Lengend Snippet: Unperturbed B cell development in Sam68 complete KO mice. A . left panel- representative images of the spleen from WT, Het (+/-) and KO (-/-) mice. Right panel- quantification of absolute splenic cell number from these mice ( n = 10–13, 4–5 independent sampling). Statistical analysis: One-way ANOVA, multiple comparison test. ** p < 0.01. B . left panel- flow cytometric analysis of B cell subpopulations in bone marrow (Hardy fractions A-F), spleen, lymph node, peritoneal cavity and T cell subpopulations in thymus from WT, Het and KO mice. Right panel- quantification of the different cell populations as depicted in the flow cytometry. For the BM B cells, % of cells are calculated as follows: Fraction A- CD43 + B220 + CD24 − BP1 − , Fraction B- CD43 + B220 + CD24 + BP1 − , Fraction C- CD43 + B220 + CD24 lo BP1 + , Fraction C´- CD43 + B220 + CD24 hi BP1 + , Fraction D -CD43 − B220 lo IgD − IgM − , Fraction E -CD43 − B220 lo IgD − IgM + and Fraction F -CD43 − B220 hi . Bars represent mean ± SEM ( n = 3 for each genotype). DP- double positive, DN- double negative

Article Snippet: Fifty nanogram of total RNA was reverse transcribed using high-capacity RNA to cDNA kit (#4387406, Thermo Fisher) and gene expression was measured by Taqman assay using the probe Khdrbs1 (Sam68) (Mm00516130_m1) and Taqman Gene expression master mix (#4369016, Thermo Fisher) following manufacturer’s protocol.

Techniques: Sampling, Comparison, Flow Cytometry

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: RNA binding protein Sam68 promotes germinal center reaction and IgG response through regulation of miR29

doi: 10.1007/s00018-026-06145-w

Figure Lengend Snippet: Impaired T-dependent immune response in Sam68 KO mice against SRBC. A . Scheme showing the experimental timeline for T-dependent SRBC immunization. B . Flow cytometric analysis of B cell activation and GC reaction in SRBC treated WT and Sam68 KO mice 10 days post immunization. C . Histogram showing expression of GL7 on the activated (CD95 + CD38 − ) B cells in the spleen of SRBC treated WT and KO mice. D . Quantification of CD38 − CD95 + activated B cells in the spleen of SRBC treated WT and KO mice. Bars represent mean ± SEM. Statistical analysis- unpaired t-test. E . Quantification of anti-SRBC IgG1, IgG2b and IgG3 in the serum of WT (black) and Sam68 KO (red) mice at day10 after immunization by flow cytometry. Statistical analysis: Two-way ANOVA, multiple comparison test ( n = 5–6, two independent experiments). * p < 0.05, ** p < 0.01, *** p < 0.001. F . Flow cytometric analysis of T-follicular helper (TfH, CD4 + CXCR5 + PD1 + ) cells in the spleen of WT and Sam68 KO mice 10 days after SRBC immunization. G. Histogram showing the expression of BCL6 in the TfH cells in WT and Sam68 KO mice. H - I . Quantification of CD4 + T cells (H) and TfH cells (I) in the spleen of immunized WT and Sam68 KO mice. Bars represent mean ± SEM. Statistical analysis- unpaired t-test

Article Snippet: Fifty nanogram of total RNA was reverse transcribed using high-capacity RNA to cDNA kit (#4387406, Thermo Fisher) and gene expression was measured by Taqman assay using the probe Khdrbs1 (Sam68) (Mm00516130_m1) and Taqman Gene expression master mix (#4369016, Thermo Fisher) following manufacturer’s protocol.

Techniques: Activation Assay, Expressing, Flow Cytometry, Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: RNA binding protein Sam68 promotes germinal center reaction and IgG response through regulation of miR29

doi: 10.1007/s00018-026-06145-w

Figure Lengend Snippet: Impaired T-dependent immune response and memory response in Sam68 complete KO mice against TNP-Ova. A. Scheme showing the experimental timeline for primary immune response using TNP-Ova. B. Quantification of total serum IgG in WT (black) and KO (red) mice at day0 (unfilled) and day10 (filled) after TNPOva immunization. The amount of serum IgG is normalized with respect to the IgG at day0. Bars represent mean ± SEM (n=5-8, total two independent experiments). Statistical analysis: One-way ANOVA, multiple comparison test. C-D- Quantification of anti-TNP IgG (C) and anti-TNP IgM (D) in the serum of TNP-Ova treated WT (black) and KO (red) mice. Statistical analysis: Two-way ANOVA, multiple comparison test. E. Flow cytometric analysis of B cell activation and GC reaction in TNP-Ova treated WT and KO mice. F. Histogram showing expression of GL7 on the activated (CD95+ CD38-) B cells in the spleen of TNP-Ova treated WT and KO mice. G. Quantification of GL7+ cells in the spleen of control and TNP-Ova treated WT (black unfilled and shaded respectively) and KO (red unfilled and shaded respectively) mice. H. Scheme showing the experimental timeline for secondary immune response using TNP-Ova. I-J. Quantification of total serum IgG (I) and anti-TNP IgG (J) in WT (black) and Sam68 KO (red) mice at different time point after TNP-Ova immunization. The amount of serum IgG is normalized with respect to the IgG at day0. Statistical analysis: Two-way ANOVA, multiple comparison test (n=5-7, two independent experiments). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

Article Snippet: Fifty nanogram of total RNA was reverse transcribed using high-capacity RNA to cDNA kit (#4387406, Thermo Fisher) and gene expression was measured by Taqman assay using the probe Khdrbs1 (Sam68) (Mm00516130_m1) and Taqman Gene expression master mix (#4369016, Thermo Fisher) following manufacturer’s protocol.

Techniques: Comparison, Activation Assay, Expressing, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: RNA binding protein Sam68 promotes germinal center reaction and IgG response through regulation of miR29

doi: 10.1007/s00018-026-06145-w

Figure Lengend Snippet: Analysis of SRBC induced GC response in competitive bone marrow chimeric Rag −/− γc −/− mice. A . Flow cytometric analysis showing the percentage of CD45.1 + WT and CD45.2 + Sam68 KO B cells in total spleen (CD19 + , left most panel) and in GC cells (CD38 − CD95 + GL7 + , right most panel). B . Quantification of the percentage of WT and KO B cells in blood, spleen and GC ( n = 9, total three independent experiments). C . Flow cytometric analysis showing the percentage of CD45.1 + WT and CD45.2 + WT B cells in blood and GC. D . Quantification of the percentage of CD45.1 + WT and CD45.2. + WT B cells in blood and GC ( n = ). Bars represent mean ± SEM. Statistical analysis: One-way ANOVA, Holm-Sidak’s multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Fifty nanogram of total RNA was reverse transcribed using high-capacity RNA to cDNA kit (#4387406, Thermo Fisher) and gene expression was measured by Taqman assay using the probe Khdrbs1 (Sam68) (Mm00516130_m1) and Taqman Gene expression master mix (#4369016, Thermo Fisher) following manufacturer’s protocol.

Techniques: Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: RNA binding protein Sam68 promotes germinal center reaction and IgG response through regulation of miR29

doi: 10.1007/s00018-026-06145-w

Figure Lengend Snippet: Sam68 regulates Traf4 expression in B cells through mir29. A - B . Real time quantitative PCR (RT-qPCR) analysis measuring expression of miR29a, b and c (A) and Traf4 (B) in WT (black bars) and Sam68 KO (red bars) splenic B cells at different time points after CD40 stimulation. Bars represent mean ± SEM ( n = 3–4 independent experiments). Statistical analysis: Two-way ANOVA, multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001. C . Flow cytometric analysis showing the electroporation efficiency of empty vector (EV) and pR miR29a in WT B cells as detected by DsRed/mRFP expression. % of DsRed or mRFP. + cells are indicated in the figure. D - E . RT-qPCR analysis measuring expression of miR29a (D) and Traf4 (E) in WT B cells electroporated either with EV or pR miR29a (29a OE). Bars represent mean ± SEM ( n = 3). Statistical analysis: one sample t-test. * p < 0.05. F . Representative western blot showing Traf4 expression in WT B cells overexpressing miR29a (29a OE) compared to EV expressing cells. Traf4 over-expressing WT B cells (Traf4 OE) are used as positive control. Lower band- endogenous Traf4, upper band- 3X FLAG tagged overexpressed Traf4 (see methods and Supplementary Fig. for details). Gapdh is used as loading control. G. Quantification of the band intensities of the western blot shown in F. Bars represent mean ± SEM ( n = 3). Statistical analysis: One way ANOVA, multiple comparison test. * p < 0.05. H. Flow cytometric analysis showing the retroviral transduction efficiency of EV and mTraf4 in WT and Sam68 KO B cells as detected by DsRed expression. % of DsRed + cells are indicated in the figure. I. Proliferation analysis of WT and Sam68 KO B cells overexpressing either EV (upper panel) or Traf4 protein (bottom panel) 48 h post stimulation with IL4 + anti-CD40 antibody. Green histograms indicate the different generation of cells as modeled by proliferation modeling in Flowjo. J. Quantification of proliferation parameters for WT and KO cells overexpressing EV (open bars) or Traf4 (T4, dotted bars). Bars represent mean ± SEM ( n = 3–4). Statistical analysis: One way ANOVA, multiple comparison test. * p < 0.05, ** p < 0.01

Article Snippet: Fifty nanogram of total RNA was reverse transcribed using high-capacity RNA to cDNA kit (#4387406, Thermo Fisher) and gene expression was measured by Taqman assay using the probe Khdrbs1 (Sam68) (Mm00516130_m1) and Taqman Gene expression master mix (#4369016, Thermo Fisher) following manufacturer’s protocol.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Comparison, Electroporation, Plasmid Preparation, Western Blot, Positive Control, Control, Retroviral, Transduction

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: RNA binding protein Sam68 promotes germinal center reaction and IgG response through regulation of miR29

doi: 10.1007/s00018-026-06145-w

Figure Lengend Snippet: Sam68 KO B cells show proliferation defect upon Anti-CD40 antibody stimulation in vitro. A . Schematic representation of the in vitro splenic B cell proliferation assay. B . Flow cytometric analysis of splenic B cell proliferation 96 h after treatment with IL4 + LPS (upper panel) and IL4 + anti-CD40 antibody (bottom panel) for WT and Sam68 KO mice. C . Quantification of proliferating B cells in response to IL4, IL4 + LPS and IL4 + anti-CD40 antibody in culture derived from WT (black) and KO (red) mice spleen ( n = 3). D . Quantification of WT and Sam68 KO B cells class switched to IgG1 in response to IL4, IL4 + LPS and IL4 + anti-CD40 antibody. Statistical analysis: Two-way ANOVA, multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Fifty nanogram of total RNA was reverse transcribed using high-capacity RNA to cDNA kit (#4387406, Thermo Fisher) and gene expression was measured by Taqman assay using the probe Khdrbs1 (Sam68) (Mm00516130_m1) and Taqman Gene expression master mix (#4369016, Thermo Fisher) following manufacturer’s protocol.

Techniques: In Vitro, Proliferation Assay, Derivative Assay, Comparison

Journal: eLife

Article Title: Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly

doi: 10.7554/eLife.85165

Figure Lengend Snippet: Western blot analysis of endogenous SAM68 expression in siRNA-transfected cells with densitometric quantification indicated below (N=3). Statistics: p-values: **** <0.0001. Figure 1—figure supplement 1—source data 1. Western blot uncropped membranes.

Article Snippet: The SAM68 WT lentivirus expression vector was generated by insertion of a FLAG-tagged SAM68 coding sequence into pLenti-CMV-MCS-GFP-SV-puro plasmid (gift from Paul Odgren Addgene plasmid # 73582) between XbaI and MluI restriction sites.

Techniques: Western Blot, Expressing, Transfection

Journal: eLife

Article Title: Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly

doi: 10.7554/eLife.85165

Figure Lengend Snippet: ( A ) HUVECs plated on FN-coated coverslips for 20 min were stained for SAM68, F-actin, cortactin and phospho-tyrosine. Scale bars=10 μm. Dotted squares depict enlarged areas (10 μm wide) shown in the same panel. ( B ) Labelling of SAM68 and FAK was performed on HUVECs plated on FN-coated coverslips for the indicated times; dotted squares depict enlarged areas shown in the same panel.

Article Snippet: The SAM68 WT lentivirus expression vector was generated by insertion of a FLAG-tagged SAM68 coding sequence into pLenti-CMV-MCS-GFP-SV-puro plasmid (gift from Paul Odgren Addgene plasmid # 73582) between XbaI and MluI restriction sites.

Techniques: Staining

Journal: eLife

Article Title: Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly

doi: 10.7554/eLife.85165

Figure Lengend Snippet: ( A ) Scheme of the experimental procedure used to induce and image artificial adhesion sites in contact with FN-coated beads. ( B ) Labelling of SAM68 and vinculin was performed 20 min after seeding cells on FN-coated beads. Dotted squares in top panels depict enlarged z-projections shown in the middle panel. Orthogonal views are shown in bottom panels. Scale bars=5 μm. ( C ) Immunolabeling of α5β1 and activated FAK (pFAK-Y397) were performed 20 min after deposition of FN-coated beads onto siCTRL- or siSAM68-transfected cells and pFAK-Y397 foci were quantified (n=at least 8 beads per condition, N=3). ( D ) siSAM68-transfected cells were transduced with lentiviral constructions encoding SAM68 WT and mutants shown in the left panel of the figure. Immunolabeling of activated FAK (pFAK-Y397) was performed 20 min after deposition of FN-coated beads onto cells and pFAK-Y397 foci were quantified (n=at least 5 beads per condition, N=4). Statistics: p-values: *<0.05 **<0.01. Student’s t-test (paired CTRL-siSAM68) was used for ( C, D ). Figure 3—source data 1. Quantification of pFAK397 foci from . Figure 3—source data 2. Quantification of pFAK397 foci from .

Article Snippet: The SAM68 WT lentivirus expression vector was generated by insertion of a FLAG-tagged SAM68 coding sequence into pLenti-CMV-MCS-GFP-SV-puro plasmid (gift from Paul Odgren Addgene plasmid # 73582) between XbaI and MluI restriction sites.

Techniques: Immunolabeling, Transfection, Transduction

Journal: eLife

Article Title: Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly

doi: 10.7554/eLife.85165

Figure Lengend Snippet: ( A ) smRNA FISH and SAM68 stainings were performed 20 min after deposition of FN-coated beads. Scale bars top image=10 μm, enlarged area 5 μm. Arrowheads point to overlapping signals between β-actin mRNA and SAM68 protein. ( B ) smRNA FISH of β-actin performed on cells 20 min after addition of FN-coated beads to cultures of siCTRL- or siSAM68-transfected cells (n=at least 12 beads per condition, N=3). Scale bars = left 10 μm, enlarged area 5 μm. ( C ) smiRNA FISH staining of β-actin performed on cells 20 min after deposition of FN-coated beads onto endothelial cells transfected with CTRL or blocking oligonucleotides (#SBE1 and #SBE2, as indicated) (n=at least 12 beads per condition, N=3). Statistics: p-values: *<0.05 **<0.01. Student’s t-test (paired CTRL-siSAM68) was used. Figure 4—source data 1. Quantification of pFAK397 foci from . Figure 4—source data 2. Quantification of pFAK397 foci from .

Article Snippet: The SAM68 WT lentivirus expression vector was generated by insertion of a FLAG-tagged SAM68 coding sequence into pLenti-CMV-MCS-GFP-SV-puro plasmid (gift from Paul Odgren Addgene plasmid # 73582) between XbaI and MluI restriction sites.

Techniques: Transfection, Staining, Blocking Assay

Journal: eLife

Article Title: Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly

doi: 10.7554/eLife.85165

Figure Lengend Snippet: ( A ) Immunofluorescence staining of α5β1 integrin was performed to identify and quantify fibrillar adhesion in siRNA transfected cells plated overnight on glass coverslips (n=at least 15; N=3). ( B ) Immunofluorescence staining of FN was performed on siRNA transfected cells plated on glass coverslips and quantification on whole-coverslip scans is expressed as the ratio of FN-stained area to the number of cells (N=8). Representative 40x field views. ( C ) Representative high magnification images of FN staining are shown with areas between the dotted lines selected for fluorescence intensity profiles. ( D ) Western blot analysis of cell-associated FN in siRNA transfected cells with densitometric quantification indicated below (N=3). ( E ) qPCR analysis of total FN (tFN) and Extra Domain-containing isoform expression in siRNA transfected cells using the indicated qPCR primer pairs (N=7). ( F ) Measurements of Luciferase activity driven by the FN1 promoter when SAM68 is overexpressed (N=5). ( G ) DNA fragments located in the FN1 promoter were quantified by qPCR in anti-SAM68 or IgG immunoprecipitated complexes (N=3). Statistics: p-values: *<0.05 **<0.01 ***<0.001 ****<0.0001. Student’s t-test (paired CTRL-siSAM68) was used for ( A, B, D, E, F ). Statistical analysis of fold enrichment in ( G ) was performed with R using pairwise t-test with p-values adjusted using ‘Bonferroni correction’. Figure 5—source data 1. Quantification of endothelial fibrillar adhesions. Figure 5—source data 2. Western blot uncropped membranes. Figure 5—source data 3. Quantification of FN1 mRNA levels. Figure 5—source data 4. Quantification of FN1 promotor reporter activity. Figure 5—source data 5. Quantification of SAM68 protein recruitment onto the FN1 promotor.

Article Snippet: The SAM68 WT lentivirus expression vector was generated by insertion of a FLAG-tagged SAM68 coding sequence into pLenti-CMV-MCS-GFP-SV-puro plasmid (gift from Paul Odgren Addgene plasmid # 73582) between XbaI and MluI restriction sites.

Techniques: Immunofluorescence, Staining, Transfection, Fluorescence, Western Blot, Expressing, Luciferase, Activity Assay, Immunoprecipitation

Journal: eLife

Article Title: Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly

doi: 10.7554/eLife.85165

Figure Lengend Snippet: Migration of individual siRNA-transfected cells was analyzed by time lapse microscopy. ( A ) Representation of individual tracks of the experiment presented in for the second siRNA directed against SAM68. ( B ) Additional quantification of average speed and total distance travelled are shown (N=3). Statistics: p-values: *<0.05. Student’s t-test (paired CTRL-siSAM68) was used.

Article Snippet: The SAM68 WT lentivirus expression vector was generated by insertion of a FLAG-tagged SAM68 coding sequence into pLenti-CMV-MCS-GFP-SV-puro plasmid (gift from Paul Odgren Addgene plasmid # 73582) between XbaI and MluI restriction sites.

Techniques: Migration, Transfection, Time-lapse Microscopy

Journal: eLife

Article Title: Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly

doi: 10.7554/eLife.85165

Figure Lengend Snippet: Distribution of average sprout length per bead observed in the 3D angiogenesis assay presented in . Results from two SAM68-targeting siRNAs in four different experiments are shown (N1 to N4).

Article Snippet: The SAM68 WT lentivirus expression vector was generated by insertion of a FLAG-tagged SAM68 coding sequence into pLenti-CMV-MCS-GFP-SV-puro plasmid (gift from Paul Odgren Addgene plasmid # 73582) between XbaI and MluI restriction sites.

Techniques: Angiogenesis Assay

Journal: eLife

Article Title: Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly

doi: 10.7554/eLife.85165

Figure Lengend Snippet: Upon integrin activation, a cytoplasmic fraction of SAM68 participates transiently in adhesion complex stabilization, through regulation of integrin signaling and localization of β-actin mRNA. In the nucleus, SAM68 concomitantly regulates the expression of key subendothelial matrix genes, thereby promoting basement membrane assembly and conditioning. These coalescent functions of SAM68 enhance endothelial cell adaptation to their microenvironment and point to an important role for SAM68 during angiogenesis.

Article Snippet: The SAM68 WT lentivirus expression vector was generated by insertion of a FLAG-tagged SAM68 coding sequence into pLenti-CMV-MCS-GFP-SV-puro plasmid (gift from Paul Odgren Addgene plasmid # 73582) between XbaI and MluI restriction sites.

Techniques: Activation Assay, Expressing, Membrane

Journal: eLife

Article Title: Coalescent RNA-localizing and transcriptional activities of SAM68 modulate adhesion and subendothelial basement membrane assembly

doi: 10.7554/eLife.85165

Figure Lengend Snippet:

Article Snippet: The SAM68 WT lentivirus expression vector was generated by insertion of a FLAG-tagged SAM68 coding sequence into pLenti-CMV-MCS-GFP-SV-puro plasmid (gift from Paul Odgren Addgene plasmid # 73582) between XbaI and MluI restriction sites.

Techniques: Transduction, Sequencing, Control, Blocking Assay

Journal: Virology Journal

Article Title: Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections

doi: 10.1186/s12985-015-0452-8

Figure Lengend Snippet: Sam68 redistribution from the nucleus to the cytoplasm. Two different FMDV-susceptible cell lines (LFBK-αvβ6, left ; and IBRS2, right ) were mock-infected or infected with FMDV at a MOI of 10 and fixed at 5 hpi. Cells were examined by IFM probing with rabbit polyclonal anti-Sam68 followed by goat-anti-rabbit-AF488 (green) and mouse monoclonal anti-FMDV VP1 followed by goat-anti-mouse-AF568 ( red ). Nuclei were stained with DAPI ( blue )

Article Snippet: Sam68 expression plasmids pGEX-2 T Sam68 (Containing GST-tagged Sam68), pcDNA3 HA-tagged Sam68-WT and pcDNA3 HA-tagged Sam68 delta-KH (Sam68-KH-del) were purchased from Addgene Cambridge, MA, USA.

Techniques: Infection, Staining

Journal: Virology Journal

Article Title: Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections

doi: 10.1186/s12985-015-0452-8

Figure Lengend Snippet: FMDV-induced cytoplasmic Sam68 co-localizes with TIA-1. LFBK cells were mock-infected or infected with FMDV at a MOI of 10 and were fixed at 3 and 5 hpi. Cells were examined by IFM probing with rabbit polyclonal anti-Sam68 followed by goat-anti-rabbit-AF488 (green) and goat polyclonal anti-TIA-1 ( a ) or mouse monoclonal anti-G3BP ( b ) followed by donkey-anti-goat-AF568 ( red , a ) or goat-anti-mouse-AF568 ( red ; b ). Nuclei were stained with DAPI ( blue )

Article Snippet: Sam68 expression plasmids pGEX-2 T Sam68 (Containing GST-tagged Sam68), pcDNA3 HA-tagged Sam68-WT and pcDNA3 HA-tagged Sam68 delta-KH (Sam68-KH-del) were purchased from Addgene Cambridge, MA, USA.

Techniques: Infection, Staining

Journal: Virology Journal

Article Title: Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections

doi: 10.1186/s12985-015-0452-8

Figure Lengend Snippet: Sam68 interacts with FMDV IRES 4. a Cartoon diagram in the upper panel describes the modular structure of FMDV A24 IRES and the location of two unpaired UAAA and a CAAA sequence motifs in domain 4 and 3, respectively. Sam68 potential binding sites are shown as a grey incomplete oval. Lower panel in Fig. 3a shows anti-Sam68 Western blot (rabbit anti-Sam68) of pull-down experiments conducted between Sam68 and selected IRES domains. IRES domains used in the experiment are shown. b Determination Sam68 binding to FMDV IRES RNAs by EMSA. WT probe in the left panel consists of 5′ biotin labeled 65 nt long synthetic RNA representing residues 435–499 of FMDV A24-Cru IRES that spanned at least 20 bases upstream and downstream of the two UAAA sequence motifs present in IRES domain 4. The binding of Sam68 to RNA probe was carried out in the presence of 100-fold excess of tRNA. The concentrations of Sam68 used are indicated in each lane. In the mutant probe in the right panel, the two UAAA motifs aremutated to UACG. c Upper panel depicts cartoon representation of the wild-type and KH-domain deleted Sam68 constructs. Lower panel shows EMSA results with the addition of Sam68-WT (left) and Sam68-delta KH (right). Probe and conditions used were the same as in section ( b ). d Determination of binding interference by various 5′ NTR RNA segments on the complexes formed between WT probe representing partial FMDV IRES domain 4 and Sam68. The binding of Sam68 to WT probe was performed under similar conditions as mentioned in section ( b ) but using a 2 μM Sam68 and 30 nM of probe. WT probe-Sam68 binding was competed with 10-fold molar excess of either full-length IRES (lane 3) or miscellaneous RNAs, including the FMDV cre (lane 4), S-fragment (lane 5), and IRES domains 2, 3, 4 (lanes 6, 7, 8, respectively). Lane 1 contains the binding mixture of Sam68 and domain 4 RNA in the absence of competitor RNAs, whereas lane 9 contains a probe alone control. Lane 2 was left blank

Article Snippet: Sam68 expression plasmids pGEX-2 T Sam68 (Containing GST-tagged Sam68), pcDNA3 HA-tagged Sam68-WT and pcDNA3 HA-tagged Sam68 delta-KH (Sam68-KH-del) were purchased from Addgene Cambridge, MA, USA.

Techniques: Sequencing, Binding Assay, Western Blot, Labeling, Mutagenesis, Construct, Control

Journal: Virology Journal

Article Title: Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections

doi: 10.1186/s12985-015-0452-8

Figure Lengend Snippet: Oligonucleotides used in this study

Article Snippet: Sam68 expression plasmids pGEX-2 T Sam68 (Containing GST-tagged Sam68), pcDNA3 HA-tagged Sam68-WT and pcDNA3 HA-tagged Sam68 delta-KH (Sam68-KH-del) were purchased from Addgene Cambridge, MA, USA.

Techniques: Sequencing

Journal: Virology Journal

Article Title: Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections

doi: 10.1186/s12985-015-0452-8

Figure Lengend Snippet: Effect of Sam68-depletion on FMDV protein and RNA synthesis using cell-free extracts. a Depletion of Sam68 from BHK-21 CFE. BHK-21 CFE was prepared as described in Materials and Methods. The depletion of Sam68 was confirmed by Western blot probing of non-depleted ( left lane ) and depleted ( right lane ) extract with anti-Sam68. b Determination of the effect of Sam68-6H addition on the translation of FMDV A 24 -Cru. FMDV A 24 -Cru RNA was translated using non-depleted or depleted BHK-21 CFE that were supplemented with 0 or 1 μM Sam68-6H as marked. The reaction was carried out at 32 ° C for 2 h and the products were resolved by SDS-PAGE and Western blot probed for FMDV 3D pol . c Determination of the effect of Sam68-6H addition on the synthesis of FMDV A 24 -Cru RNA. FMDV A 24 -Cru RNA was used for RNA synthesis using non-depleted or depleted BHK-21 CFE that were supplemented with 0–2.5 μM Sam68-6H as marked. The reaction was carried out at 37 ° C for 5 h and the products were SDS-PAGE resolved by dot blotting

Article Snippet: Sam68 expression plasmids pGEX-2 T Sam68 (Containing GST-tagged Sam68), pcDNA3 HA-tagged Sam68-WT and pcDNA3 HA-tagged Sam68 delta-KH (Sam68-KH-del) were purchased from Addgene Cambridge, MA, USA.

Techniques: Western Blot, SDS Page

Journal: Virology Journal

Article Title: Analysis of the interaction between host factor Sam68 and viral elements during foot-and-mouth disease virus infections

doi: 10.1186/s12985-015-0452-8

Figure Lengend Snippet: Sam68 interacts with FMDV 3C pro and 3D pol . a Co-immunoprecipitation of FMDV 3D pol and Sam68 during FMDV infection. BHK-21 cells either mock-infected or infected with FMDV at a MOI of 10 were lysed and the lysates were immunoprecipitated using either anti-FMDV 3D pol or anti-Sam68 and the eluates examined by Western blot. One lane in each panel (as indicated below) was not subject to IP as a control. Equal amount of isotype control antibody served as an IP control. The eluates from the anti-3D pol IP reaction were probed with anti-Sam68 (left panel). Conversely, the Sam68 IP eluates were probed with an anti-3D pol (right panel). In the left panel, lane 1 corresponds to the isotype IP control, lane 2 is mock-infected cell lysate (1:10 dilution), lane 3 is a FMDV-infected cell lysate (1:10 dilution) that was not IP and lane 4 is the anti-3D pol IP eluate from FMDV-infected cell lysates. Similarly, in the right panel, lane 1 corresponds to the isotype control, lane 2 is mock-infected cell lysate (1:10 dilution), lane 3 is a FMDV-infected cell lysate (1:10 dilution) that was not IP, and lane 4 is the anti-Sam68 IP eluate from FMDV-infected cell lysates. b The fragments (frag) listed in the table correspond to the amino acid (aa) sequence of FMDV 3D pol , starting from the N-terminus: frag #1 aa 1–48, frag #2 aa 49–108, frag #3 aa 109–157, frag #4 aa 158–217, frag #5 aa 218–268, frag #6 aa 269–331, frag #7 aa 332–404, and frag #8 aa 405–470. A scrambled peptide was used as a negative control. c Computational prediction of the interaction between FMDV 3D pol and Sam68. (i) Electrostatic surface representation of FMDV 3D pol in the docking pose (PDB: 1U09); red color depicts the negatively charged surface, white shows the neutral surface, and blue color shows the positively charged surface. Color intensity is proportional to the surface charge. Areas under dashed lines indicate Sam68 binding interface of FMDV 3D pol . (ii) Electrostatic surface representation of Sam68 in the docking pose. Surface charge and color annotation are same as section (i). Surface marked with dashed lines indicates FMDV 3D pol binding interface of Sam68. (iii) Electrostatic representation of Sam68 docked to FMDV 3D pol . FMDV 3D pol green docked on Sam68 blue in cartoon representation. The 3D pol frag-4 residues 193–217 (orange), frag-5 residues 221, 222, 225, 226 (magenta) and frag-8 residues 453–470 (red) form the Sam68 binding interface of 3D pol ( d ) LFBK cells were uninfected or infected with FMDV at a MOI of 10, and cells were harvested at 1, 3, and 5 hpi by treatment with versine. Left panel: cell lysates were IP with mouse monoclonal anti-FMDV 3C pro , and examined by Western blot probing with rabbit polyclonal anti-Sam68 (N-terminus). Right panel: collected cells were lysed and separated into nuclear and cytoplasmic fractions, and the cytoplasmic fractions were examined by Western blot probing with rabbit polyclonal anti-Sam68 (N-terminus). Loading control is indicated confirming equivalent loading per lane

Article Snippet: Sam68 expression plasmids pGEX-2 T Sam68 (Containing GST-tagged Sam68), pcDNA3 HA-tagged Sam68-WT and pcDNA3 HA-tagged Sam68 delta-KH (Sam68-KH-del) were purchased from Addgene Cambridge, MA, USA.

Techniques: Immunoprecipitation, Infection, Western Blot, Control, Sequencing, Negative Control, Binding Assay

Journal: PLoS ONE

Article Title: Signal-Regulated Pre-mRNA Occupancy by the General Splicing Factor U2AF

doi: 10.1371/journal.pone.0001418

Figure Lengend Snippet: (A) Immunoblot analysis of GST pull-down reactions involving recombinant Sam68 and GST-fusion proteins of U2AF65 and U2AF35. The GST protein served as a control. Immunodetection with anti-Sam68 and anti-GST antibodies is shown in the upper and lower panels, respectively. Black bars on the left indicate positions of marker bands. (B) Co-immunoprecipitation from LB17-lymphoma cell lysates with an anti-Sam68 antibody or a corresponding control antibody (CoAb). In lane 3, the lysate was loaded. Precipitates were analysed by immunoblotting with an anti-U2AF65 antibody. Arrows indicate U2AF65 band; asterisks denote bands caused by immunoglobulins. (C) Co-immunoprecipitation experiment as in (B), in the presence (+) or absence (−) of RNase A. Symbols are as in (B).

Article Snippet: Cells were lysed in CoIP buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.5% NP40; supplemented with 0.1 mM DTT; 1 mM PMSF; 2 μg/ml Aprotinin and 2 μg/ml Leupeptin) and the cleared lysate was incubated with a polyclonal rabbit anti-Sam68 antibody (C20, Santa Cruz Biotechnology) bound to protein A/G agarose at 4°C for 1h.

Techniques: Western Blot, Recombinant, Control, Immunodetection, Marker, Immunoprecipitation

Journal: PLoS ONE

Article Title: Signal-Regulated Pre-mRNA Occupancy by the General Splicing Factor U2AF

doi: 10.1371/journal.pone.0001418

Figure Lengend Snippet: (A) Scheme indicating positions of the Sam68 binding sites (black oval and box, respectively) in CD44 exon v5 (open box) and the upstream intron (line). The Sam68 consensus binding site in the wild-type RNA sequence used for the electrophoretic mobility shift assay (EMSA) in panel B is boxed. The mutated nucleotide of the A/C mutant is indicated in red. (B) EMSA using recombinant Sam68 and radioactively labeled RNA oligonucleotide probes comprising either the wild-type or the A/C-mutant version of the intronic Sam68 binding site (see panel A). (C) v5-luciferase fusion activity from LB17 lymphoma cells transfected with different pETv5luc splice-reporter genes . They were mutated for either the exonic (L/CC mutant ), the intronic (A/C mutant), or both Sam68 binding sites (double mutant). Cells were treated with 12-o-tetradecanoylphorbol-13-acetate (TPA, 40 ngml −1 ) (+) or with DMSO (solvent control) (−) for 6h prior to lysis and measurement of luciferase activity. Error bars indicate standard deviations from three independent transfections.

Article Snippet: Cells were lysed in CoIP buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.5% NP40; supplemented with 0.1 mM DTT; 1 mM PMSF; 2 μg/ml Aprotinin and 2 μg/ml Leupeptin) and the cleared lysate was incubated with a polyclonal rabbit anti-Sam68 antibody (C20, Santa Cruz Biotechnology) bound to protein A/G agarose at 4°C for 1h.

Techniques: Binding Assay, Sequencing, Electrophoretic Mobility Shift Assay, Mutagenesis, Recombinant, Labeling, Luciferase, Activity Assay, Transfection, Solvent, Control, Lysis

Journal: PLoS ONE

Article Title: Signal-Regulated Pre-mRNA Occupancy by the General Splicing Factor U2AF

doi: 10.1371/journal.pone.0001418

Figure Lengend Snippet: (A) Immunoblotting of non-phosphorylated (−) and ERK-phosphorylated (P) recombinant Sam68 preparations used in (B). Phosphorylation of Sam68 at ERK target sites results in a molecular weight shift (arrow) . Black bar on the left indicates marker band. (B) Electrophoretic mobility shift assay using RNA oligonucleotide probes comprising the exonic or intronic Sam68 binding sites and different amounts of either non-phosphorylated (−) or phosphorylated (P) recombinant Sam68.

Article Snippet: Cells were lysed in CoIP buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.5% NP40; supplemented with 0.1 mM DTT; 1 mM PMSF; 2 μg/ml Aprotinin and 2 μg/ml Leupeptin) and the cleared lysate was incubated with a polyclonal rabbit anti-Sam68 antibody (C20, Santa Cruz Biotechnology) bound to protein A/G agarose at 4°C for 1h.

Techniques: Western Blot, Recombinant, Phospho-proteomics, Molecular Weight, Marker, Electrophoretic Mobility Shift Assay, Binding Assay

Journal: PLoS ONE

Article Title: Signal-Regulated Pre-mRNA Occupancy by the General Splicing Factor U2AF

doi: 10.1371/journal.pone.0001418

Figure Lengend Snippet: (A) Detection of Sam68 binding to an endogenous CD44 pre-mRNA fragment (spanning the v5 exon and 100 bp upstream intron sequence) in LB17 lymphoma cells by RNP immunoprecipitations. A control antibody (CoAb) or an anti-Sam68 antibody were used for precipitations. Lower panels show amplifications from the lysate used as input for the precipitations. Bands correspond to the expected size of 228 bp. Positions of primers used for amplification are indicated as arrowheads in scheme on top; Sam68 binding sites (black oval and box, respectively), CD44 exons v4 and v5 (open boxes), intron (line). –RT represents reactions without reverse transcriptase. (B) RNP immunoprecipitations with either a control antibody (CoAb) or an anti-Sam68 antibody from lysates of LB17 lymphoma cells that were transfected with the pETv5 minigene construct . The cells were either left untreated (−) or treated with 40 ng/ml of 12-o-tetradecanoylphorbol-13-acetate (TPA) for 10 min. A fragment from the minigene pre-mRNA spanning the v5 exon and 450 bp upstream intron sequence (In-v5) was amplified using the primers indicated (scheme on top; symbols as in panel A). Specific amplification of the minigene pre-mRNA is achieved by the forward primer that is complementary to the deletion junction of the v4 exon in the construct. To check for potential differential loss of material during the precipitation and washing procedure, we monitored the amounts of GAPDH mRNA unspecifically bound to the antibody and/or the agarose beads (lower panel). Bands correspond to the expected sizes of 600 bp (In-v5) and 278 bp (GAPDH). All PCR amplifications were in linear phase as verified with different amounts of cDNA.

Article Snippet: Cells were lysed in CoIP buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.5% NP40; supplemented with 0.1 mM DTT; 1 mM PMSF; 2 μg/ml Aprotinin and 2 μg/ml Leupeptin) and the cleared lysate was incubated with a polyclonal rabbit anti-Sam68 antibody (C20, Santa Cruz Biotechnology) bound to protein A/G agarose at 4°C for 1h.

Techniques: Binding Assay, Sequencing, Control, Amplification, Reverse Transcription, Transfection, Construct

Journal: PLoS ONE

Article Title: Signal-Regulated Pre-mRNA Occupancy by the General Splicing Factor U2AF

doi: 10.1371/journal.pone.0001418

Figure Lengend Snippet: (A) Schematic drawing of the examined CD44 minigene pre-mRNA region (CD44 exon v5, open box; upstream intron, line; Sam68 binding sites, black oval and box, respectively; arrows, PCR primer). (B) RNP immunoprecipitations with an anti-U2AF65 antibody or a control antibody (CoAb) from lysates of LB17 lymphoma cells that were transfected with the pETv5 minigene construct containing CD44 exon v5. Cells were left either untreated (−) or treated with phorbol ester (TPA). TPA treatment and RNP immunoprecipitations were performed as described in legend to . (C) RNP immunoprecipitations of U2AF65 from LB17 lymphoma cells co-transfected with the pETv5 minigene construct and either a Sam68 expression plasmid (+) or the empty expression vector as a control (−). Bands correspond to the expected sizes of 600 bp (In-v5) and 278 bp (GAPDH). All PCR amplifications were in linear phase as verified with different amounts of cDNA.

Article Snippet: Cells were lysed in CoIP buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.5% NP40; supplemented with 0.1 mM DTT; 1 mM PMSF; 2 μg/ml Aprotinin and 2 μg/ml Leupeptin) and the cleared lysate was incubated with a polyclonal rabbit anti-Sam68 antibody (C20, Santa Cruz Biotechnology) bound to protein A/G agarose at 4°C for 1h.

Techniques: Binding Assay, Control, Transfection, Construct, Expressing, Plasmid Preparation

Journal: Molecular cell

Article Title: The Sam68 STAR RNA-binding protein regulates mTOR alternative splicing during adipogenesis.

doi: 10.1016/j.molcel.2012.02.007

Figure Lengend Snippet: Figure 1. Sam68–/– Mice Are Leaner Than Their Wild-Type Littermate Controls (A) Sam68/ or Sam68+/+ mice were maintained on a normal chow diet and weighed at 6, 12, and 24 weeks of age, as indicated. The bar denotes the mean and significance as determined by Student’s t test. **p < 0.001 and *p < 0.01. (B) The fat pads were isolated from Sam68/ or Sam68+/+ mice and weighed. EF, epididymal fat; IF, inguinal fat; RF, retroperitoneal fat; BF, brown fat; H, heart. Error bars represent ± SEM. (C) Representative transverse MRI images are shown for mice 15–16 or 28 weeks of age. The percentage body WAT is indicated. (D) The food intake for Sam68/ or Sam68+/+ mice was calculated as daily average values adjusted to body weight. Error bars represent ± SEM.

Article Snippet: Histology and Immunoblotting Mice were perfused with 4% paraformaldehyde, and WAT was processed for paraffin sections stained with hematoxylin and eosin or oil red O. RNA Interference Mouse KHDRBS1 (Sam68) siGENOME SMARTpool siRNA (catalog number M-065115-01-0010) and siGFP siRNAwere ordered fromDharmacon (Thermo Scientific).

Techniques: Isolation

Journal: Molecular cell

Article Title: The Sam68 STAR RNA-binding protein regulates mTOR alternative splicing during adipogenesis.

doi: 10.1016/j.molcel.2012.02.007

Figure Lengend Snippet: Figure 2. Sam68–/– Mice Are Protected from Dietary-Induced Obesity (A and B) Sam68/ or Sam68+/+ mice were fed either a chow diet (A) or a high-fat diet (HFD) (B) for 16 weeks. The weight of the mice is indicated and expressed as the mean body weight. Error bars represent ± SEM. (C) Mice fed a HFD were starved overnight and then given orally a glucose bolus (dose), and blood glucose was then monitored with time. Error bars represent ± SEM. (D) Insulin tolerance tests (ITTs) were performed on anesthetised males by intraperitoneal administration of insulin, and blood glucose was monitored with time. Error bars represent ± SEM. (E) Mice fed a HFD were starved overnight and then challenged with insulin for 5 min. Activation of the insulin receptor (IR) was assessed by immunoblotting liver extracts with anti-pY1162/3 IR antibodies. Total IR serves as a loading control.

Article Snippet: Histology and Immunoblotting Mice were perfused with 4% paraformaldehyde, and WAT was processed for paraffin sections stained with hematoxylin and eosin or oil red O. RNA Interference Mouse KHDRBS1 (Sam68) siGENOME SMARTpool siRNA (catalog number M-065115-01-0010) and siGFP siRNAwere ordered fromDharmacon (Thermo Scientific).

Techniques: Activation Assay, Western Blot, Control

Journal: Molecular cell

Article Title: The Sam68 STAR RNA-binding protein regulates mTOR alternative splicing during adipogenesis.

doi: 10.1016/j.molcel.2012.02.007

Figure Lengend Snippet: Figure 3. Sam68 Is Required for Adipogenesis of 3T3-L1 Cells (A) 3T3-L1 cells were transfected with siGFP (control) or siSam68. 3T3-L1 cells or siSam68-treated 3T3-L1 cells were transfected with pcDNA3.1 (control) or an expression vector encoding hSam68. Adipocyte differentiation was assessed by oil red O staining. (B and C) 3T3-L1 cells were stably transfected with pRetrosuper or Sam68sh pRetrosuper. A bulk population of pRetrosuper and two individual clones (Sam68sh2 and Sam68sh22) were selected for analysis by immunoblotting using Sam68 and actin antibodies, and adipogenesis was analyzed as described in (A).

Article Snippet: Histology and Immunoblotting Mice were perfused with 4% paraformaldehyde, and WAT was processed for paraffin sections stained with hematoxylin and eosin or oil red O. RNA Interference Mouse KHDRBS1 (Sam68) siGENOME SMARTpool siRNA (catalog number M-065115-01-0010) and siGFP siRNAwere ordered fromDharmacon (Thermo Scientific).

Techniques: Transfection, Control, Expressing, Plasmid Preparation, Staining, Stable Transfection, Clone Assay, Western Blot

Journal: Molecular cell

Article Title: The Sam68 STAR RNA-binding protein regulates mTOR alternative splicing during adipogenesis.

doi: 10.1016/j.molcel.2012.02.007

Figure Lengend Snippet: Figure 4. Sam68 Inactivation Increases the Retention of Intron 5 of mTOR mRNA during Adipogenesis (A) Splicing of mTOR exon 4–6 schematic and the intron 5 retention leading to the generation of mTORi5. (B) The mRNA levels of mTORi5, mTORexon45-46, PPARg, and Sam68 normalized to PRMT1 as assessed by RT-qPCR from control pRetrosuper (pRetrosuper) and Sam68 knockdown (Sam68sh) 3T3-L1cells at 0 and 4 days of differentiation. Error bars represent ± SEM. (C) Schematic of the mTOR minigene and its validation. Detection, by RT-PCR, of mTORexon4-6, mTORi5, and mTORexon4 in wild-type MEFs after knockdown of Sam68 (siSam68) or overexpression of Sam68 (myc-Sam68). mTORexon4 and GAPDH serve as internal expression controls.

Article Snippet: Histology and Immunoblotting Mice were perfused with 4% paraformaldehyde, and WAT was processed for paraffin sections stained with hematoxylin and eosin or oil red O. RNA Interference Mouse KHDRBS1 (Sam68) siGENOME SMARTpool siRNA (catalog number M-065115-01-0010) and siGFP siRNAwere ordered fromDharmacon (Thermo Scientific).

Techniques: Quantitative RT-PCR, Control, Knockdown, Biomarker Discovery, Reverse Transcription Polymerase Chain Reaction, Over Expression, Expressing

Journal: Molecular cell

Article Title: The Sam68 STAR RNA-binding protein regulates mTOR alternative splicing during adipogenesis.

doi: 10.1016/j.molcel.2012.02.007

Figure Lengend Snippet: Figure 5. Sam68 Associates with RNA Sequences within mTOR Intron 5 (A) Schematic of the mTOR minigene in its original form followed by the different mutations in the Sam68-binding sequences previously identified. (B) Sequence of intron 5 SBS#1 and SBS#2, as well as the mutated (UUUCA) versions. Peptide pull-down assays were performed using 3T3-L1 cells and immunoblotted with Sam68 antibodies. (C) ChIP analysis using anti-Sam68 or control IgGs. Bound DNA was analyzed in triplicate by qPCR with the indicated primers specific for the mTOR gene. Mean values are expressed as fold enrichment. Error bars represent ± SEM. (D) Minigene assays show the requirement for SBS#1 and SBS#2. Fold inclusion of intron 5 is expressed as the level of mTORi5 over mTORexon4 and normalized with the total level of the minigene expression product (mTORexon4). Error bars represent ± SEM.

Article Snippet: Histology and Immunoblotting Mice were perfused with 4% paraformaldehyde, and WAT was processed for paraffin sections stained with hematoxylin and eosin or oil red O. RNA Interference Mouse KHDRBS1 (Sam68) siGENOME SMARTpool siRNA (catalog number M-065115-01-0010) and siGFP siRNAwere ordered fromDharmacon (Thermo Scientific).

Techniques: Binding Assay, Sequencing, Control, Expressing

Journal: Molecular cell

Article Title: The Sam68 STAR RNA-binding protein regulates mTOR alternative splicing during adipogenesis.

doi: 10.1016/j.molcel.2012.02.007

Figure Lengend Snippet: Figure 6. Sam68-Deficient Cells Exhibit Decreased mTORC1 and mTORC2 Activity (A) Cellular extracts were immunoblotted with the indicated antibodies during adipocyte differentiation. Tubulin and S6 served as loading controls, and the molecular markers are shown on the left in kilodaltons (kDa). The relative quantification is shown below the panels. (B) pRetrosuper or Sam68sh2 3T3-L1 cells display impaired mTORC1 and mTORC2 activity following insulin stimulation. Starved cells (16 hr) were left untreated or treated with rapamycin and/or insulin 15 min prior to stimulation. Cell extracts were prepared and immunoblotted with the indicated antibodies. (C) Sam68sh 3T3-L1 cells were transiently transfected with empty vector or an mTOR expression vector and induced to differentiate for 6 days. Transfections were repeated 1 day after differentiation initiation and adipogenesis visualized by oil red O staining. (D) Quantification of percentage of cells positive for oil red O staining. The graph is representative of >10 fields. Error bars represent ± SEM.

Article Snippet: Histology and Immunoblotting Mice were perfused with 4% paraformaldehyde, and WAT was processed for paraffin sections stained with hematoxylin and eosin or oil red O. RNA Interference Mouse KHDRBS1 (Sam68) siGENOME SMARTpool siRNA (catalog number M-065115-01-0010) and siGFP siRNAwere ordered fromDharmacon (Thermo Scientific).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Expressing, Staining