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: Antibody , anti-SAM68 (rabbit polyclonal) , Sigma-Aldrich , Cat # HPA051280 , IF (1:200) WB (1:200).

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: Antibody , anti-SAM68 (rabbit polyclonal) , Sigma-Aldrich , Cat # HPA051280 , IF (1:200) WB (1:200).

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 ) 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: Antibody , anti-SAM68 (rabbit polyclonal) , Sigma-Aldrich , Cat # HPA051280 , IF (1:200) WB (1:200).

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: Antibody , anti-SAM68 (rabbit polyclonal) , Sigma-Aldrich , Cat # HPA051280 , IF (1:200) WB (1:200).

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: Antibody , anti-SAM68 (rabbit polyclonal) , Sigma-Aldrich , Cat # HPA051280 , IF (1:200) WB (1:200).

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: Antibody , anti-SAM68 (rabbit polyclonal) , Sigma-Aldrich , Cat # HPA051280 , IF (1:200) WB (1:200).

Techniques: Activation Assay, Expressing

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: Antibody , anti-SAM68 (rabbit polyclonal) , Sigma-Aldrich , Cat # HPA051280 , IF (1:200) WB (1:200).

Techniques: Transduction, Sequencing, Blocking Assay

Journal: Cell reports

Article Title: A Dynamic Splicing Program Ensures Proper Synaptic Connections in the Developing Cerebellum.

doi: 10.1016/j.celrep.2020.107703

Figure Lengend Snippet: Figure 5. Sam68 Preferentially Represses Exons Characterized by a Weak Branchpoint 5368289352488500(A and B) Branchpoint score (A) and GC content (B) for up- and downregulated exons in Sam68KO cerebella with respect to non-regulated exons (Wilcoxon rank-sum test). (C) Distribution of the DPSI of exons differentially regulated between Sam68KO and WT cerebella. Whiskers indicate 1.5 interquartile range. Welch’s t test; ****p < 0.0001. (D and E) Left panels: scheme of the AS region of the Arhgef9 (D) and Arhgap12 (E) gene, with regulated (red) and constitutive exons (green) and qPCR primers (arrows) and the splicing events in WT and Sam68KO cerebella. Shown is RT-PCR analysis for Sam68-regulated events in Arhgef9 (D) and Arhgap12 (E) with PSI values from densitometric analysis (mean ± SEM, n = 6, paired t test). Right panels: bar graphs showing qPCR analyses of Sam68 CLIPs for Arhgef9 (D) and Arhgap12 (E) pre-mRNAs in P10 mouse cerebella. Immunoglobulin G (IgG) was used as a control (mean ± SEM, n = 5, two tailed paired t test, *p < 0.05). (F and G) qPCR analyses of U2AF65 CLIPs for Arhgef9 (F) and Arhgap12 (G) pre-mRNAs in WT and Sam68KO P10 cerebella (mean ± SEM, n = 5, multiple Student’s t test, *p < 0.05). (H) Western blot analysis of U2AF65 and Sam68 expression in WT and Sam68KO P10 mouse cerebella. See also Figure S5.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-Sam68 Bethyl Laboratories Cat#A302-110A; RRID:AB_1604289 Donkey whole antibody anti Rabbit IgG HRP-linked GE Healthcare Cat# NA934; RRID: AB_772206 Sheepwhole antibody anti mouse IgGHRPlinked GE Healthcare Cat# NA931; RRID:AB_772210 Alexa Fluor-488 goat anti mouse Thermo Fisher Scientific Cat# A11001; RRID:AB_2534069 Alexa Fluor-594 goat anti-rabbit Thermo Fisher Scientific Cat# A11037; RRID:AB_2534095 Mouse monoclonal anti 5-bromo-20deoxyuridine (BrdU) Millipore Cat# 05-633, RRID:AB_11212826 Mouse monoclonal anti CALB1 Sigma-Aldrich Cat# C9848, RRID:AB_476894 Mouse monoclonal anti-U2AF65 Sigma-Aldrich Cat#U4758, RRID:AB_262122 Rabbit polyclonal anti VGLUT1 Synaptic System Cat# 135 303, RRID:AB_887875 Mouse monoclonal anti-ACTIN Santa Cruz Cat# sc-47778, RRID: AB_626632 Chemicals, Peptides, and Recombinant Proteins Dnase I from bovine pancreas Sigma Aldrich Cat# DN25; CAS 9003-98-9 Trypsin from bovine pancreas Sigma Aldrich Cat#T8003 Poly-L-lysine Sigma Aldrich Cat# P9155 Trypsin inhibitor from bovine pancreas Sigma Aldrich Cat#T9003 5-Bromo-20-deoxyuridine Sigma Aldrich Cat#B5002 B27tm Supplement Thermo Fisher Scientific,GIBCO Cat#17504-044 (+)-Bicuculline Tocris Cat#0130 Tetrodotoxin Tocris Cat#1078 RNAprotect Tissue Reagent QIAGEN Cat# 76104 Tissue-Tek O.C.T.

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

Journal: Cancer cell

Article Title: ILF2 Is a Regulator of RNA Splicing and DNA Damage Response in 1q21-Amplified Multiple Myeloma

doi: 10.1016/j.ccell.2017.05.011

Figure Lengend Snippet: shRNA Knockdown and Overexpression Experiments

Article Snippet: Rabbit polyclonal anti-Sam68 , Santa Cruz , Cat# sc-333.

Techniques: shRNA, Knockdown, Over Expression, Immunoprecipitation, Immunofluorescence, Virus, Plasmid Preparation, Microarray, Recombinant, Reverse Transcription, Fractionation, Mutagenesis, Software