Journal: Journal of Clinical Investigation

Article Title: uPAR isoform 2 forms a dimer and induces severe kidney disease in mice

doi: 10.1172/jci124793

Figure Lengend Snippet: Figure 7. Blocking Src activity reduces proteinuria in msuPAR2-Tg mice. (A) Glomerular Src activity was determined by immunofluorescent staining of the kidney cryosections with p-Src monoclonal antibody (green). Podocin was used as a podocyte marker (red). Shown are the representative glomeruli with p-Src immunofluorescent staining. As indicated by the p-Src staining intensity, glomerular Src phosphorylation was readily observed in the kidney sections from msuPAR2-Tg, but not from other investigated mice. Scale bars: 20 μm. (B) Treatment with Src inhibitors lowered proteinuria in msuPAR2-Tg mice. Both PP1 and Src inhibitor 1 (5 mg/kg) were administered into the randomly grouped msuPAR2-Tg mice intraperitoneally, with vehicle controls receiving the same amount of DMSO. n = 8 Src inhibitor group; n = 7 vehicle control group. At 72 hours after treatment, proteinuria was significantly reduced by Src inhibitor treatment. Two-way ANOVA; data were log-transformed to normal distribution. **P < 0.01.

Article Snippet: Src phosphorylation was labeled with p–c-Src antibody (A96, 1:100, Santa Cruz Biotechnology Inc.).

Techniques: Blocking Assay, Activity Assay, Staining, Marker, Phospho-proteomics, Control, Transformation Assay

Journal: Journal of Clinical Investigation

Article Title: uPAR isoform 2 forms a dimer and induces severe kidney disease in mice

doi: 10.1172/jci124793

Figure Lengend Snippet: Figure 8. Glomerular c-Src activity is increased in human FSGS kidney. Immunofluorescent staining with p–c-Src antibody was performed for the frozen sections of deidentified human kidney biopsies. Synaptopodin was used as a podocyte marker. Shown is a representative of 4 batches of immu- nostaining. While a minimal amount of c-Src phosphorylation was observed in the glomeruli of healthy donors (n = 3), glomerular p–c-Src intensity was increased in 6 out of 10 FSGS patients. Overlap of p–c-Src (green) and synpo (red ) indicates that p–c-Src was local- ized in podocytes. Of note, only 11 out of 24 observed glomeruli were positive for p–c-Src, from which 64% were focal, 36% globally but not evenly. In contrast, the increase of pSrc was not observed in other glomerular diseases, including SLE (n = 2), MPGN (n = 2), and MCD (n = 4). Scale bar: 20 μm. NT, normal kidney tissue.

Article Snippet: Src phosphorylation was labeled with p–c-Src antibody (A96, 1:100, Santa Cruz Biotechnology Inc.).

Techniques: Activity Assay, Staining, Marker, Phospho-proteomics

Journal: Journal of Cell Science

Article Title: A unique role for clathrin light chain A in cell spreading and migration

doi: 10.1242/jcs.224030

Figure Lengend Snippet: CLCa depletion significantly inhibits spreading-induced signaling. (A) siRNA-transfected cells held in suspension for 1 h (left panel) or plated on collagen IV-coated dishes for the indicated times (minutes, right panel) were lysed and subjected to western blotting with anti-active FAK [pFAK(Y397)], active Src [pSrc(Y416)] and anti-phosphorylated paxillin [pPax(Y118)] antibodies. (B) Protein phosphorylation in control cells at 30 min after plating was set as 100%. The results represent a summary from five to seven experiments. *P<0.05; **P<0.01. (C) Lysates from plated cells treated as in A were analyzed by western blotting with antibodies against Src-dependent FAK phosphorylation sites (Y576 and Y925). The blots shown represent one of three independent experiments.

Article Snippet: Antibodies against the following proteins were used: CLCa (1:1000, sc-28276), CLCb (1:500, sc-376414), actin (1:1000, sc-1616) from Santa Cruz Biotechnology, FAK (1:2000, 610088) and β1-integrin (1:1000, 610467) from BD Transduction Labs, phosphorylated FAK(Y397) (1:1000, 44-624G), phosphorylated paxillin(Y118) (1:1000, 44-722G) from Fisher Scientific, Src (1:2000, 2108), phosphorylated Src(Y416) (1:1000, MAB2685, 2101), phosphorylated FAK(Y576) (1:1000, 3281), FAK(Y925) (1:1000, 3284) from Cell Signaling, phosphorylated Src(Y416) (1:1000, MAB2685) from RD Systems, WAVE1/Scar (1:1000, 07-037), Rac1 (1:2000, 05-389) from Millipore.

Techniques: Transfection, Western Blot

Journal: Breast Cancer Research : BCR

Article Title: JMJD6 is a driver of cellular proliferation and motility and a marker of poor prognosis in breast cancer

doi: 10.1186/bcr3200

Figure Lengend Snippet: JMJD6 expression and prognosis in intrinsic subtypes of breast cancer . Box plots of JMJD6 expression demonstrate (A) higher JMJD6 probeset intensity (log2) in ER - tumors compared with ER + tumors, with P < 0.001 by Mann-Whitney Rank Sum test; and (B) highest JMJD6 probeset intensity (log2) in claudin-low and basal subtypes, followed by HER2 -enriched and LumB subtypes, and lowest in LumA subtypes. The Dunn method was used to perform pairwise multiple comparison among the subtypes. * P < 0.05 between a pair of subtypes. Dots of boxplots (A, B) represent outliers in the 90 th and 10 th percentiles. Kaplan-Meier survival curves based on below-median (low) and above-median (high) JMJD6 expression are shown for (C) ER + patients, (D) patients of LumA subtype, (E) patients of LumB subtype, and (F) ER + , tamoxifen-treated patients. The numbers in the parentheses equal patient numbers in each group, and the log rank P value is indicated at the bottom left of each figure.

Article Snippet: Anti-JMJD6 monoclonal antibody (Santa Cruz, Santa Cruz, CA, USA: Sc-28348) was used at a dilution of 1:50 along with antigen retrieval by heat and Tris-EDTA (pH 9.1).

Techniques: Expressing, MANN-WHITNEY, Comparison

Journal: Breast Cancer Research : BCR

Article Title: JMJD6 is a driver of cellular proliferation and motility and a marker of poor prognosis in breast cancer

doi: 10.1186/bcr3200

Figure Lengend Snippet: Hazard ratios of JMJD6 expression groups in various subtypes of breast cancer

Article Snippet: Anti-JMJD6 monoclonal antibody (Santa Cruz, Santa Cruz, CA, USA: Sc-28348) was used at a dilution of 1:50 along with antigen retrieval by heat and Tris-EDTA (pH 9.1).

Techniques: Expressing

Journal: Breast Cancer Research : BCR

Article Title: JMJD6 is a driver of cellular proliferation and motility and a marker of poor prognosis in breast cancer

doi: 10.1186/bcr3200

Figure Lengend Snippet: Nuclear JMJD6 protein expression is associated with high-grade breast tumors . Representative breast cancer cores from tissue microarray that were stained with JMJD6 antibody are shown. Low-grade (left panel), intermediate-grade (middle panel), and high-grade breast tumors (right panel) are shown (magnification, ×60). The table in the figure shows univariate analysis of JMJD6 and clinical parameters.

Article Snippet: Anti-JMJD6 monoclonal antibody (Santa Cruz, Santa Cruz, CA, USA: Sc-28348) was used at a dilution of 1:50 along with antigen retrieval by heat and Tris-EDTA (pH 9.1).

Techniques: Expressing, Microarray, Staining

Journal: Breast Cancer Research : BCR

Article Title: JMJD6 is a driver of cellular proliferation and motility and a marker of poor prognosis in breast cancer

doi: 10.1186/bcr3200

Figure Lengend Snippet: Expression of JMJD6 enhances proliferation . (A) WST-1 assays using MCF-7-J1-OE clones (J1-C2, J1-C3, and J1-C7) showed increased proliferation over vector control cells (Vec). SiRNA-mediated knockdown of JMJD6 and resultant decrease in proliferation is shown in (B) MCF-7 and MDA-MB231. Four individual siRNAs (A, B, C, and D) were used: Sc, scrambled siRNA; and NT, non-transfected cells served as control. Inset in each panel shows protein levels of JMJD6 after siRNA transfection. β-actin immunoblots show equal protein loading in all lanes.

Article Snippet: Anti-JMJD6 monoclonal antibody (Santa Cruz, Santa Cruz, CA, USA: Sc-28348) was used at a dilution of 1:50 along with antigen retrieval by heat and Tris-EDTA (pH 9.1).

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

Journal: Breast Cancer Research : BCR

Article Title: JMJD6 is a driver of cellular proliferation and motility and a marker of poor prognosis in breast cancer

doi: 10.1186/bcr3200

Figure Lengend Snippet: JMJD6 promotes motility in breast cancer cell lines . (A) Scatter phenotype was scored based on three levels of scattering (compact, loose, scatter) (upper panel). Bar graphs represent quantification of scattering of colonies by using the Student t test (lower panel). MCF-7 J1 OE clones had a higher percentage of scattered or loosely scattered clones than did the control Vec cells. (B) Wound-healing assay showed that MCF-7 J1 clones displayed higher motility in comparison to Vec (upper panel); quantification of wound closure as a ratio to the Vec is shown in the lower panel. * P ≤ 0.05 by Student t test in comparison to Vec control. (C) Boyden-chamber assay results of MDA-MB231 cells with JMJD6 siRNA-mediated knockdown were normalized to fold change in proliferation at Day 2 of WST-1 measurement. Two of three siRNA knockdowns of JMJD6 showed significantly decreased motility, and all siRNA knockdowns of JMJD6 showed decreased invasiveness. * P ≤ 0.05; ** P ≤ 0.001 by Student's t- test when compared with Sc siRNA.

Article Snippet: Anti-JMJD6 monoclonal antibody (Santa Cruz, Santa Cruz, CA, USA: Sc-28348) was used at a dilution of 1:50 along with antigen retrieval by heat and Tris-EDTA (pH 9.1).

Techniques: Clone Assay, Control, Wound Healing Assay, Comparison, Boyden Chamber Assay, Knockdown

Journal: Breast Cancer Research : BCR

Article Title: JMJD6 is a driver of cellular proliferation and motility and a marker of poor prognosis in breast cancer

doi: 10.1186/bcr3200

Figure Lengend Snippet: IPA analysis of JMJD6-regulated genes showed enrichment in cell-cycle function . (A) The heatmap (top panel) shows that hierarchic clustering of the genes changed after siRNA knockdown and overexpression of JMJD6 . The sample labels are on the top of the heat map. Two clusters representing JMJD6-induced and JMJD6-repressed genes were selected (middle heat-map panel) and subjected to IPA analysis. (B) Table shows a list of the top 10 functions that are enriched in cells with altered levels of JMJD6.

Article Snippet: Anti-JMJD6 monoclonal antibody (Santa Cruz, Santa Cruz, CA, USA: Sc-28348) was used at a dilution of 1:50 along with antigen retrieval by heat and Tris-EDTA (pH 9.1).

Techniques: Knockdown, Over Expression

Journal: Breast Cancer Research : BCR

Article Title: JMJD6 is a driver of cellular proliferation and motility and a marker of poor prognosis in breast cancer

doi: 10.1186/bcr3200

Figure Lengend Snippet: JMJD6 represses TGF-β2 expression . RT-qPCR assays of TGF-βs after JMJD6 siRNA-mediated knockdown are shown in (A) MCF-7 and (B) MDA-MB231. Consistently, T GF-β2 and TGF-β1 mRNA levels were upregulated on JMJD6 knockdown. (C) Immunoblots showed that the amount of secreted TGF-β2 protein in conditioned media was higher after JMJD6 siRNA-mediated knockdown in MCF-7 and MDA-MB231. (D) RT-qPCR assay showed that MCF7 J1 OE clones had a lower level of TGF-β1 and TGF-β2 transcripts than did the Vec cells. (E) Western blot showed a dramatic decrease in secreted TGF-β2 protein, in the MCF-7 J1 clones, as compared with the Vec cells. NS, a Ponceau-stained blot of the conditioned media to demonstrate protein loading. Student's t - test was performed by using scrambled siRNA versus JMJD6 siRNA and Vec cells versus MCF-7J1 clones. * P ≤ 0.05; ** P ≤ 0.005.

Article Snippet: Anti-JMJD6 monoclonal antibody (Santa Cruz, Santa Cruz, CA, USA: Sc-28348) was used at a dilution of 1:50 along with antigen retrieval by heat and Tris-EDTA (pH 9.1).

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Western Blot, Clone Assay, Staining

Journal: Breast Cancer Research : BCR

Article Title: JMJD6 is a driver of cellular proliferation and motility and a marker of poor prognosis in breast cancer

doi: 10.1186/bcr3200

Figure Lengend Snippet: JMJD6 expression affects SMAD phosphorylation . Immunoblots show that the levels of total SMAD2/3, phosphorylated SMAD2, and phosphorylated SMAD3 are decreased in MCF-7 J1-OE clones and increased in JMJD6 siRNA-mediated knockdown in MCF-7 and MDA-MB231. The numbers next to the SMAD2P/3P denote the expected sites of phosphorylation detected by the antibodies. NS, nonspecific bands from the blots, which indicate equal loading of the protein lysates.

Article Snippet: Anti-JMJD6 monoclonal antibody (Santa Cruz, Santa Cruz, CA, USA: Sc-28348) was used at a dilution of 1:50 along with antigen retrieval by heat and Tris-EDTA (pH 9.1).

Techniques: Expressing, Phospho-proteomics, Western Blot, Clone Assay, Knockdown

Journal: Breast Cancer Research : BCR

Article Title: JMJD6 is a driver of cellular proliferation and motility and a marker of poor prognosis in breast cancer

doi: 10.1186/bcr3200

Figure Lengend Snippet: TGF-β2 inhibits proliferation in MCF-7 . (A) Immunoblots showed that treatment of MCF-7 by recombinant human (rh) TGF-β2 (5 ng/ml) resulted in enhanced Smad2 phosphorylation, which could be effectively nullified by SB431542. (B) Treatment of JMJD6 siRNA-transfected MCF-7 cells with SB431542 (10 μ M ) resulted in rescue of the proliferation defect. Ratio of OD at Day 3 to Day 1 in JMJD6 knockdown cells was normalized to the scrambled siRNA control. * P ≤ 0.05; ** P ≤ 0.005 between DMSO and SB431542 treatments. (C) Treatment of MCF-7 J1-OE and Vec cells with rhTGF-β2 resulted in decreased proliferation, as measured by WST-1 assay. ** P ≤ 0.001 between BSA and rhTGF-β2 treatments.

Article Snippet: Anti-JMJD6 monoclonal antibody (Santa Cruz, Santa Cruz, CA, USA: Sc-28348) was used at a dilution of 1:50 along with antigen retrieval by heat and Tris-EDTA (pH 9.1).

Techniques: Western Blot, Recombinant, Phospho-proteomics, Transfection, Knockdown, Control, WST-1 Assay

Journal: Breast Cancer Research : BCR

Article Title: JMJD6 is a driver of cellular proliferation and motility and a marker of poor prognosis in breast cancer

doi: 10.1186/bcr3200

Figure Lengend Snippet: Decrease of cyclin E2 protein level in JMJD6 knockdown . Western blot detection of G 1 cell-cycle cyclins is shown. A decrease in cyclin E2 (CCNE2) level with decrease in JMJD6 levels was evident in MCF-7 and MDA-MB231. Cyclin E1 (CCNE1) and cyclin D1 (CCND1) displayed an inconsistent change on JMJD6 siRNA transfection in both MCF-7 and MDA-MB231 cells.

Article Snippet: Anti-JMJD6 monoclonal antibody (Santa Cruz, Santa Cruz, CA, USA: Sc-28348) was used at a dilution of 1:50 along with antigen retrieval by heat and Tris-EDTA (pH 9.1).

Techniques: Knockdown, Western Blot, Transfection

Journal: Breast Cancer Research : BCR

Article Title: JMJD6 is a driver of cellular proliferation and motility and a marker of poor prognosis in breast cancer

doi: 10.1186/bcr3200

Figure Lengend Snippet: Clinical association of JMJD6 with TGF-β2 . Pearson correlation of clinical expression of JMJD6 with TGF-β2 suggests a negative correlation with TGF-β2 ( P = 9.13 × 10 -8 ). Scatterplot shows the normalized JMJD6 expression of each patient sample with the corresponding normalized TGF-β2 expression in the clinical dataset of 2,034 breast cancer patients.

Article Snippet: Anti-JMJD6 monoclonal antibody (Santa Cruz, Santa Cruz, CA, USA: Sc-28348) was used at a dilution of 1:50 along with antigen retrieval by heat and Tris-EDTA (pH 9.1).

Techniques: Expressing

Journal: RNA

Article Title: ZFC3H1 and U1-70K promote the nuclear retention of mRNAs with 5′ splice site motifs within nuclear speckles

doi: 10.1261/rna.079104.122

Figure Lengend Snippet: ZFC3H1 depletion leads to cytoplasmic accumulation of endogenous 5′SS motif containing mRNAs (or intronic polyadenylated transcripts). ( A ) Workflow for RNA Frac-seq. ZFC3H1- or control-depleted U2OS cells were fractionated into nuclear and cytoplasmic/ER fractions (see Materials and Methods for more details). RNA was purified from these fractions and from total cell lysates, and then analyzed by Illumina sequencing. ( B ) Nuclear “N” and cytoplasmic “C” fractions were collected from ZFC3H1- or control-depleted U2OS cells, then separated by SDS-PAGE and analyzed by immunoblot for nuclear (Aly), ER (Trap-α), and cytoplasmic (tubulin) protein markers. ( C ) Lysates collected from ZFC3H1- or control-depleted U2OS cells (96 h post-transduction with lentiviral-delivered ZFC3H1-2 shRNA) were analyzed by immunoblot for ZFC3H1 and tubulin. ( D ) Fold change in total levels of intronic polyadenylated (IPA) transcripts (ZFC3H1 depletion vs. control depletion) ( x -axis), plotted against the change in the total levels of fully processed mRNA (using cUTR reads, y -axis). Each dot corresponds to reads from one gene that is known to produce IPA transcripts (listed in Supplemental Table 1 ). Note that ZFC3H1 depletion leads to the up-regulation of IPA transcripts, but not fully processed mRNAs. ( E ) ( Top ) Schematic of a fully processed mRNA and IPA transcript generated from the same gene. Note that the IPA transcript is generated from a 3′ cleavage/polyadenylation signal in the first intron and contains a 5′SS motif. ( Bottom ) genome browser tracks of the PCF11 gene in control- “shCon” or ZFC3H1-depleted “shZFC” cells at 500× and 50× resolution. Note the large peak for the IPA transcript (intronic cleavage/polyadenylation site is denoted with a red arrow), which is up-regulated in ZFC3H1-depleted cells. Also note that the reads corresponding to the full-length transcript are unaffected by ZFC3H1 depletion. ( F ) Similar to D , except that the fold change of IPA transcript levels in the cytoplasmic fraction (ZFC3H1 depletion vs. control depletion, x -axis) is plotted against the fold change in the nuclear fraction (ZFC3H1 depletion vs. control depletion, y -axis). Note that ZFC3H1 depletion leads to cytoplasmic accumulation of many IPA transcripts (compare blue dots to red). To account for reads from fully processed mRNAs, the IPA transcript levels are normalized to the cUTR transcript levels of the same gene. ( G ) Similar to E , except the IPA peaks in the nuclear and cytoplasmic fractions are shown for PCF11 . In control-depleted cells, reads from the PCF11 IPA are enriched in the nuclear, but not the cytoplasmic fractions. In ZFC3H1-depleted cells, reads from the PCF11 IPA are at comparable levels in the nuclear and cytoplasmic fractions. ( H – J ) Genome browser tracks of three IPA transcript-producing genes, “ CCDC71 ,” “ BRD3 ,” and “ ZFPM1 .” Note the accumulation of IPA transcripts in the cytoplasmic fractions upon ZFC3H1 depletion. The intronic 3′ cleavage/polyadenylation sites are denoted by red arrows.

Article Snippet: Antibodies used in this study include rabbit polyclonals against ZFC3H1 (also known as CCDC131) (Bethyl Laboratories, A301-457A), MTR4 (also known as SKIV2L2) (Bethyl Laboratories, A300-614A), PABPN1 (Bethyl Laboratories, A303-523A), U1-70K (Abcam, ab83306), Aly , and TRAPα ( ) or mouse monoclonals against U1-70K (Sigma-Aldrich, clone 9C4.1), mAb414 (Sigma), SC35 (Clone SC35, Sigma), and α-tubulin (DM1A, Sigma).

Techniques: Control, Purification, Illumina Sequencing, SDS Page, Western Blot, Transduction, shRNA, Generated

Journal: RNA

Article Title: ZFC3H1 and U1-70K promote the nuclear retention of mRNAs with 5′ splice site motifs within nuclear speckles

doi: 10.1261/rna.079104.122

Figure Lengend Snippet: ZFC3H1 is required for the nuclear retention of 5′SS motif containing mRNAs. ( A ) U2OS cells were treated with different lentivirus shRNAs against ZFC3H1 (“ZFC3H1-1” and “ZFC3H1-2”), MTR4 (“MTR4-1 + 2”) or control shRNA. Lysates were collected 96 h post-transduction, separated by SDS-PAGE and immunoprobed for ZFC3H1, MTR4, or tubulin. Note that to effectively deplete MTR4, cells were treated with lentivirus containing two shRNA plasmids. ( B ) Schematic of the intronless ( Δi ) ftz reporter ( ftz-Δi ) construct used in this study, with and without the V5-His element in the 3′UTR ( ftz-Δi-5 ′ SS ). Note that the V5-His element contains a consensus 5′SS motif, which promotes nuclear retention. ( C , D ) Control-, MTR4-, and ZFC3H1-depleted cells were transfected with the intronless ftz reporter plasmid (± 5 ′ SS ). Eighteen to twenty-four hours later, the cells were fixed and the mRNA was visualized by FISH. Note that depletion of ZFC3H1, but not MTR4, caused the cytoplasmic accumulation of the ftz-Δi-5′SS mRNA. Representative images are shown in C (scale bar, 10 µm) and quantification is shown in D . Each bar represents the average and standard error of at least three independent experiments, each experiment consisting of at least 30 to 60 cells. Student's t -test was performed for D. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001. ( E ) Schematic of CCDC71 -IPA reporter used in this study (see also H). The position of the FISH probe used to visualize the IPA RNA is marked in gray and the position of the 3′ cleavage site in the intron is as indicated. ( F ) U2OS cells were transfected with the CCDC71-IPA reporter and, 18 to 24 hours later, the cells were fixed. The IPA transcript was visualized by FISH and nuclear speckles were visualized by immunofluorescence against SC35. Representative images are shown with a merged overlay showing the CCDC71-IPA mRNA in red and SC35 in green. Scale bar, 10 µM. Examples of CCDC71-IPA /SC35 colocalization are indicated with arrows. ( G , H ) Control- and ZFC3H1-depleted cells were transfected with the CCDC71-IPA reporter and the IPA transcript was visualized by FISH. ZFC3H1 depletion increased the cytoplasmic accumulation of the CCDC71-IPA . Representative images are shown in G (scale bar, 10 µm) and quantification is shown in H . Each bar represents the average and standard error of at least three independent experiments, each experiment consisting of at least 30 to 60 cells. Student's t -test was performed for H. (**) P < 0.01. ( I – K ) ePAT assay and 3′RACE were used to examine 3′ end processing. ( I ) Schematic of the ePAT assay as described in . The ftz -specific ( F′ ) and universal ( R′ ) primers used to amplify the ePAT amplicon are indicated. The sequence of the ePAT amplicon before the cleavage site is shown in J and is 117 nt long. ( J ) The sequence of the end of the 3′UTR is shown. Indicated in bold are the ftz -specific F ′ primer annealing site (used in the ePAT and 3 ′ RACE experiments), the hexanucleotide motif, and the cleavage site (as determined by 3 ′ RACE experiments on mRNAs derived from U2OS cells transfected with either ftz-Δi or ftz-Δi-5′SS ). ( K ) PCR products from the ePAT assay were separated on a 1% agarose gel and stained with ethidium bromide. Lane 1 : Molecular weight markers with sizes in bp indicated on the left ; lanes 3–6 : ePAT amplicons from U2OS cells that were transfected with plasmids containing the indicated versions of the ftz reporter (without [ Δi ] or with [ i ] an intron, without or with the 5′SS motif). Note that the amplicons generated from all four reactions are the same length (∼230 nt). Since the amplified region in the 3 ′ UTR is 117 bp long (see J ), and the universal primer has a 14 nt extension (see J ), the poly(A)-tail is estimated to be ∼100 nt long.

Article Snippet: Antibodies used in this study include rabbit polyclonals against ZFC3H1 (also known as CCDC131) (Bethyl Laboratories, A301-457A), MTR4 (also known as SKIV2L2) (Bethyl Laboratories, A300-614A), PABPN1 (Bethyl Laboratories, A303-523A), U1-70K (Abcam, ab83306), Aly , and TRAPα ( ) or mouse monoclonals against U1-70K (Sigma-Aldrich, clone 9C4.1), mAb414 (Sigma), SC35 (Clone SC35, Sigma), and α-tubulin (DM1A, Sigma).

Techniques: Control, shRNA, Transduction, SDS Page, Construct, Transfection, Plasmid Preparation, Immunofluorescence, Amplification, Sequencing, Derivative Assay, Agarose Gel Electrophoresis, Staining, Molecular Weight, Generated

Journal: RNA

Article Title: ZFC3H1 and U1-70K promote the nuclear retention of mRNAs with 5′ splice site motifs within nuclear speckles

doi: 10.1261/rna.079104.122

Figure Lengend Snippet: ZFC3H1 and U1-70K function in the same pathway for the nuclear retention of 5′SS motif containing mRNAs. ( A ) U2OS cells were treated with lentivirus shRNA against either U1-70K, ZFC3H1, or a mixture of the two. Lysates were collected 96 h post-transduction, separated by SDS-PAGE and immunoprobed for U1-70K, ZFC3H1, and mAb414. Note that to effectively deplete U1-70K, cells were treated with lentivirus containing four shRNA plasmids. Also note that the asterisk (*) denotes a non-specific band. ( B , C ) Control-, U1-70K-, ZFC3H1-, or codepleted cells were transfected with the intronless ftz reporter ± 5′SS as described in . Note that the cytoplasmic/nuclear distribution of ftz-Δi-5′SS mRNA in cells codepleted of U1-70K and ZFC3H1 resembles the distribution in single depletion cells, suggesting that both proteins function in the same pathway. Representative images are shown in C (scale bar, 10 µm) and quantification is shown in D . Each bar represents the average and standard error of at least three independent experiments, each experiment consisting of at least 30 to 60 cells. Student's t -test was performed for C . (*) P < 0.05, (**) P < 0.01, (***) P < 0.001. ( D ) HEK cells expressing carboxy-terminally tagged ZFC3H1 (ZFC3H1-FLAG) were lysed and subjected to immunoprecipitation reactions with FLAG M2 beads or mouse IgG (“Control IP”). Immunoprecipitates were separated by SDS-PAGE and immunoprobed for FLAG and U1-70K. For comparison, 1% of the input lysate was also analyzed. The full-length ZFC3H1-FLAG protein is denoted by the asterisk (*) and a shorter band, likely a degradation product, is denoted by the pound (#) sign.

Article Snippet: Antibodies used in this study include rabbit polyclonals against ZFC3H1 (also known as CCDC131) (Bethyl Laboratories, A301-457A), MTR4 (also known as SKIV2L2) (Bethyl Laboratories, A300-614A), PABPN1 (Bethyl Laboratories, A303-523A), U1-70K (Abcam, ab83306), Aly , and TRAPα ( ) or mouse monoclonals against U1-70K (Sigma-Aldrich, clone 9C4.1), mAb414 (Sigma), SC35 (Clone SC35, Sigma), and α-tubulin (DM1A, Sigma).

Techniques: shRNA, Transduction, SDS Page, Control, Transfection, Expressing, Immunoprecipitation, Comparison

Journal: RNA

Article Title: ZFC3H1 and U1-70K promote the nuclear retention of mRNAs with 5′ splice site motifs within nuclear speckles

doi: 10.1261/rna.079104.122

Figure Lengend Snippet: ZFC3H1 and U1-70K are required for nuclear retention of 5′SS motif containing mRNAs in speckles. ( A , B ) Control or ZFC3H1-depleted U2OS cells were microinjected with plasmids containing the βG-Δi reporter ± the 5′SS motif. After the indicated times, the cells were fixed and stained for βG mRNA by FISH and for the nuclear speckle marker SC35 by immunofluorescence. ( A ) Example images of U2OS cells fixed 2 h post-injection with βG reporter mRNA ± the 5′SS motif, with each row representing a single field of view with white arrows pointing to examples of βG mRNA/SC35 colocalization. The merged overlayed image shows βG mRNA in red, SC35 in green. ( B ) Quantification of the degree of βG mRNA/SC35 colocalization in cells depleted of ZFC3H1 or control shRNA treatment by Pearson correlation coefficient analysis as previously described . Each bar represents the average and standard error of three independent experiments, each experiment consisting of 150 to 200 nuclear speckles from 15 to 20 cells. Note that ZFC3H1 is not required for the targeting of 5′SS motif containing mRNAs to nuclear speckles. ( C – E ) Control, U1-70K- or ZFC3H1-depleted U2OS cells were transfected with ftz-Δi ± 5 ′ SS motif. Eighteen to twenty-four hours post-transfection, the cells were fixed and stained for ftz mRNA by FISH and for the nuclear speckle marker SC35 by immunofluorescence. Representative images, with each row depicting a single field of view, is shown in C with merged overlays showing ftz mRNA in red and SC35 in green. Scale bar, 10 µM. Examples of ftz /SC35 colocalization are indicated with arrows. ( D ) The degree of ftz /SC35 colocalization by Pearson correlation coefficient analysis was quantified as above, except that values <0.25 were counted as “not colocalized.” Note that U1-70K or ZFC3H1 depletion leads to decreased level of colocalization between ftz mRNA and SC35. Each bar represents the average and standard error of three independent experiments, each experiment consisting of 100–200 nuclear speckles from 10 to 20 cells. ( E ) The amount of ftz reporter mRNA present in nuclear speckles as a percentage of either the nuclear (“Spec/Nuc”) or total cellular (“Spec/Total”) mRNA levels in transfected cells. Each data point represents the average and standard error of the mean of at least three independent experiments, each experiment consisting of 10–20 cells. Student's t -test was performed, (**) P < 0.01, (***) P < 0.001.

Article Snippet: Antibodies used in this study include rabbit polyclonals against ZFC3H1 (also known as CCDC131) (Bethyl Laboratories, A301-457A), MTR4 (also known as SKIV2L2) (Bethyl Laboratories, A300-614A), PABPN1 (Bethyl Laboratories, A303-523A), U1-70K (Abcam, ab83306), Aly , and TRAPα ( ) or mouse monoclonals against U1-70K (Sigma-Aldrich, clone 9C4.1), mAb414 (Sigma), SC35 (Clone SC35, Sigma), and α-tubulin (DM1A, Sigma).

Techniques: Control, Staining, Marker, Immunofluorescence, Injection, shRNA, Transfection