Journal: Scientific Reports

Article Title: Cyclin-dependent kinase 9 is required for the survival of adult Drosophila melanogaster glia

doi: 10.1038/s41598-017-07179-8

Figure Lengend Snippet: cdk9 is necessary for glial survival. ( a–h ) Representative images of 7d old adult brains immunostained with anti-Repo to label glia for Fig. 1i,j. The images are maximum projections of optical sections. The outlined area demarcates the region of the central brain where the number of glia were quantified. ( i ) Number of anti-Repo-expressing glia in the central brain of 7d old adult flies in mir-31a mutants (mir-31a KO/KO) and where UAS-GFP , UAS-CG16947 , UAS-cdk9 RNAi or UAS-p53 RNAi lines were expressed in glia using Repo-Gal4 . Glia were represented as a percentage of the control animals, Repo-Gal4 > UAS-GFP (left axis). Raw number of Repo-expressing glia in the central brain of the genotypes (right axis). One-way ANOVA was used for statistical analysis and error bars represent SEM. ( j ) Number of anti-Repo-expressing glia in the central brain of 7d old adult flies. UAS-GFP , UAS-CG16947 and UAS-cdk9 RNAi expression was driven by Repo-Gal4 under the control of tubulin Gal80 ts (tubGal80 ts ). Adult-only expression was achieved by rearing the flies at 18 °C then moving the flies to 29 °C to allow Repo-Gal4 activity after eclosion. Data is represented as a percentage of the number of glia in the UAS-GFP control condition (left axis). Raw number of Repo-expressing glia in the central brain of the genotypes (right axis). One-way ANOVA was used for statistical analysis and error bars represent SEM.

Article Snippet: 2.5 mg of protein was incubated together with 2 μg of rabbit anti-CG16947 (Lifespan Biosciences, LS-B13598 ), or 2 μg of mouse anti-cdk9 (Novus Biologicals, H00001025-M07) overnight on a tube rotator at 4 °C.

Techniques: Expressing, Control, Activity Assay

Journal: Scientific Reports

Article Title: Cyclin-dependent kinase 9 is required for the survival of adult Drosophila melanogaster glia

doi: 10.1038/s41598-017-07179-8

Figure Lengend Snippet: cdk9 is necessary for adult glial survival. ( a–d ) Representative images of 7d old adult brains immunostained with anti-Repo to label glia for Fig. 2e,f. The images are maximum projections of optical sections. The outlined area demarcates the region of the central brain where the number of glia were quantified. ( e ) Number of anti-Repo-expressing cells in the central brain of 7d old mir-31a mutants (mir-31a KO/KO) when either UAS-GFP or UAS-cdk9 was expressed only in adult glia with Repo-Gal4 under the control of tubGal80 ts . Repo-Gal4 > UAS-GFP represent otherwise wildtype control animals to demonstrate the effectiveness of CDK9 at restoring the wildtype levels of glia in the adult brain. Data is represented as a percentage of the number of glia in the UAS-GFP control condition (left axis). Raw number of Repo-expressing glia in the central brain of the genotypes (right axis). One-way ANOVA was used. Error bars represent SEM. ( f ) Number of anti-Repo-expressing cells in the central brain of 7d old adults expressing either UAS-GFP or UAS-cdk9 only in glia with Repo-Gal4 under the control of Gal80 ts . Data is represented as a percentage of the number of glia in the UAS-GF P control condition (left axis). Raw number of Repo-expressing glia in the central brain of the genotypes (right axis). Unpaired Student’s t-test was used. Error bars represent SEM.

Article Snippet: 2.5 mg of protein was incubated together with 2 μg of rabbit anti-CG16947 (Lifespan Biosciences, LS-B13598 ), or 2 μg of mouse anti-cdk9 (Novus Biologicals, H00001025-M07) overnight on a tube rotator at 4 °C.

Techniques: Expressing, Control

Journal: Scientific Reports

Article Title: Cyclin-dependent kinase 9 is required for the survival of adult Drosophila melanogaster glia

doi: 10.1038/s41598-017-07179-8

Figure Lengend Snippet: cdk9 interacts directly with CG16947. ( a ) Alignment of the Drosophila melanogaster (fly) and human protein sequences of cdk9. Highlighted portion denotes region that the commercial anti-cdk9 antibody was generated against human cdk9 protein. ( b ) CG16947 (50 kDa) was detected in the lysates from Repo-Gal4 > UAS-GFP (Repo > UAS-GFP) and Repo-Gal4 > UAS-CG16947 (Repo > UAS-CG16947) used for co-immunoprecipitation in 3d, e, f and Supplemental Fig. . Kinesin (120 kDa) was used as a loading control to ensure that similar levels of protein were loaded from both genotypes. ( c ) cdk9 (49 kDa) was detected in the lysates from Repo-Gal4 > UAS-GFP (Repo > UAS-GFP) and Repo-Gal4 > UAS-CG16947 (Repo > UAS-CG16947) used for co-immunoprecipitation in 3d, e, f and Supplemental Fig . Kinesin (120 kDa) was used as a loading control to ensure that similar levels of protein were loaded from both genotypes. ( d ) Western blot of anti-CG16947 co-immunoprecipitation. Flies were of either Repo-Gal4 > UAS-GFP (Repo > UAS-GFP) or Repo > UAS-CG16947 (Repo > UAS-CG16947) genotype. Blot was probed with anti-cdk9. Lysate refers to the input for the co-immunoprecipitation. A control condition where the beads were incubated with the anti-CG16947 antibody in the absence of lysate was done to distinguish anti-CG16947 antibody elution from the beads versus the CG16947 protein itself. A 49 kDa cdk9 band was detected in the lysates and the without DTT elution of the anti-rchy1 pulldown in both Repo-Gal4 > UAS-GFP (Repo > UAS-GFP) and Repo-Gal4 > UAS-CG16947 (Repo > UAS-CG16947) conditions. ( e ) Co-immunoprecipitation with anti-cdk9. Anti-cdk9 pulled down CG16947 protein (50 kDa). An increase in the number and intensity of the bands recognised by anti-CDK9 in the MG132-treated samples compared to when the lysate and co-immunoprecipitation was done without MG132 suggest that CG16947 was actively degraded via the proteasome in vitro . The multiple bands observed below the expected size for CG16947 likely represent degraded CG16947, which can be observed when the proteasome is inhibited in vitro . ( f ) Co-immunoprecipitation with anti-cdk9. anti-cdk9 pulled down cdk9 protein that was ubiquitinated as shown by the bands above the expected size for cdk9, 49 kDa and degraded, as shown by the bands below 49 kDa. An increase in the number and intensity of the bands recognised by anti-ubiquitin in the MG132-treated samples demonstrated that cdk9 was actively ubiquitinated and degraded via the proteasome in vitro . A higher exposure of the blot is shown in Supplemental Fig. where ubiquitinated cdk9 and its degraded products can be detected in the lysates without MG132.

Article Snippet: 2.5 mg of protein was incubated together with 2 μg of rabbit anti-CG16947 (Lifespan Biosciences, LS-B13598 ), or 2 μg of mouse anti-cdk9 (Novus Biologicals, H00001025-M07) overnight on a tube rotator at 4 °C.

Techniques: Generated, Immunoprecipitation, Control, Western Blot, Incubation, In Vitro, Ubiquitin Proteomics

Journal: Scientific Reports

Article Title: Cyclin-dependent kinase 9 is required for the survival of adult Drosophila melanogaster glia

doi: 10.1038/s41598-017-07179-8

Figure Lengend Snippet: Table depicting number of samples, mean and SEM of all genotypes tested.

Article Snippet: 2.5 mg of protein was incubated together with 2 μg of rabbit anti-CG16947 (Lifespan Biosciences, LS-B13598 ), or 2 μg of mouse anti-cdk9 (Novus Biologicals, H00001025-M07) overnight on a tube rotator at 4 °C.

Techniques:

Journal: bioRxiv

Article Title: Histone chaperone HIRA facilitates transcription elongation to regulate insulin sensitivity and obesity-associated adipose expansion

doi: 10.1101/2025.03.21.644577

Figure Lengend Snippet: 3T3-L1 white preadipocytes were infected with a lentiviral vector expressing human HIRA with C-terminal dTAG and HA double tags, followed by lentiviral CRISPR/Cas9- Hira gRNA to delete endogenous Hira . At D4 of adipogenesis, cells were treated with dTAG-13 for 6h. a , Pie charts depicting the genomic distribution of HIRA binding regions at D4. b , Heat maps were aligned around the center of HIRA binding sites on promoters (6,838), primed enhancers (1,998), active enhancers (AEs, 17,862) and other regions (1,751) at D4. c , Average binding profiles of HIRA-HA, H3K27ac, S5P-Pol II, S2P-Pol II, CDK9 and SPT6 around the center of HIRA + promoters and AEs. Normalized read counts are shown. d , Profiles of HIRA-HA, H3K27ac, S5P-Pol II, S2P-Pol II, CDK9 and SPT6 on gene bodies of HIRA + promoter-associated genes. g , ChIP–Seq profiles of HIRA-HA, H3K27ac, S5P-Pol II, S2P-Pol II, CDK9 and SPT6 were displayed on Adipoq , Mlxipl (ChREBP) and Fasn loci.

Article Snippet: Anti-S5P-Pol II (13523), anti-S2P-Pol II (13499S), anti-CDK9 (2316T) and anti-SPT6 (15616) were from Cell Signaling Technology.

Techniques: Infection, Plasmid Preparation, Expressing, CRISPR, Binding Assay, ChIP-sequencing

Journal: Nucleic Acids Research

Article Title: Patient mutation in AIRE disrupts P-TEFb binding and target gene transcription

doi: 10.1093/nar/gkr527

Figure Lengend Snippet: Mutant AIREfsx protein does not interact with P-TEFb. ( A ) Mutant AIREfsx protein does not interact with CDK9. GAL, GAL.AIRE and GAL.AIREfsx chimeras were expressed in HeLa cells. Proteins were immunoprecipitated with anti-GAL antibodies (αGAL) and the co-immunoprecipitation with CDK9 was detected with anti-CDK9 antibodies by western blotting (Top panel, IP:WB). The lower three panels contain 5% of input proteins for immunoprecipitations (WB). ( B ) Mutant AIREfsx protein cannot recruit CDK9 to DNA. Sonicated lysates from HeLa cells co-expressing GAL.AIRE or GAL.AIREfsx fusion proteins and G5HIV2SVEDA were immunoprecipitated with anti-CDK9 (αCDK9) antibodies for ChIP. Enrichment of amplicons is presented as fold enrichment over IgG control (CDK9/IgG). Black and striped bars represent values for the WT GAL.AIRE and mutant GAL.AIREfsx chimeras with SEM depicted by error bars. G5HIV2SVEDA and ChIP amplicons are depicted above and below the bar graph, respectively (promoter, Pr; 5′-end of gene, 5′; 3′-end of gene, 3′, polyadenylation signal, pA; untranscribed region, U). G5HIV2SVEDA contains five GAL binding sites (UAS) and the TATA box (T) upstream of the α-globin gene (αgl) with the inserted fragment from the fibronectin 1 gene (FN1).

Article Snippet: Antibodies used for western blotting, co-immunoprecipitation (co-IP), ChIP or immunofluorescence: anti-AIRE (sc-33188 Santa Cruz Biotechnology), anti-Flag (M2 Sigma-Aldrich), anti-CDK9 (sc-484 Santa Cruz Biotechnology, ab10874 Abcam), anti-RNAPII (ab5408 Abcam, MMS-126 R Covance), anti-S2P (ab5095 Abcam), anti-GAL (sc-510 Santa Cruz Biotechnology), anti-GAPDH (AM4300 Ambion), anti-α Tubulin (DM1A Sigma-Aldrich), anti-U5-116 kD (A300-957A Bethyl labs) rabbit and mouse control IgG (Santa Cruz Biotechnology).

Techniques: Mutagenesis, Immunoprecipitation, Western Blot, Sonication, Expressing, Binding Assay

Journal: Nucleic Acids Research

Article Title: Patient mutation in AIRE disrupts P-TEFb binding and target gene transcription

doi: 10.1093/nar/gkr527

Figure Lengend Snippet: AIRE requires CDK9 for its effects on pre-mRNA splicing. ( A ) Time course of CDK9 siRNA knockdown. HeLa cells were transfected with CDK9 or control siRNAs (cntrl). Subsequently, cells were harvested at various time-points and protein levels were determined with specific antibodies by western blotting. In the upper and lower panels, levels of CDK9 and GAPDH are presented, respectively. ( B ) Protein effectors are expressed in CDK9-knockdown cells. HeLa cells were co-transfected with CDK9 or control siRNAs. GAL or the WT GAL.AIRE chimera and G5HIV2dsx were co-expressed as indicated. Protein levels of expressed proteins are presented in the upper two panels with those of CDK9 and tubulin in the lowest panels, respectively. ( C ) CDK9 siRNA knock-down inhibits effects of AIRE on splicing. Upper panel contains a schematic representation of the G5HIV2dsx minigene with amplicons used to amplify spliced, unspliced or total dsx transcripts by RT–qPCR. Relative splicing efficiency was determined and is presented for the WT GAL.AIRE chimera (black bars) as fold activation relative to GAL (white bars) for cells transfected with CDK9 or control siRNAs. Error bars represent SEM.

Article Snippet: Antibodies used for western blotting, co-immunoprecipitation (co-IP), ChIP or immunofluorescence: anti-AIRE (sc-33188 Santa Cruz Biotechnology), anti-Flag (M2 Sigma-Aldrich), anti-CDK9 (sc-484 Santa Cruz Biotechnology, ab10874 Abcam), anti-RNAPII (ab5408 Abcam, MMS-126 R Covance), anti-S2P (ab5095 Abcam), anti-GAL (sc-510 Santa Cruz Biotechnology), anti-GAPDH (AM4300 Ambion), anti-α Tubulin (DM1A Sigma-Aldrich), anti-U5-116 kD (A300-957A Bethyl labs) rabbit and mouse control IgG (Santa Cruz Biotechnology).

Techniques: Transfection, Western Blot, Quantitative RT-PCR, Activation Assay

Journal: Nucleic Acids Research

Article Title: Patient mutation in AIRE disrupts P-TEFb binding and target gene transcription

doi: 10.1093/nar/gkr527

Figure Lengend Snippet: By recruiting P-TEFb, AIRE induces splicing of an endogenous TRA gene. ( A ) Schematic representation of the KRT14 gene with primer pairs used to amplify spliced and unspliced transcripts by RT–qPCR. ( B ) WT AIRE and mutant AIREfsx proteins are expressed in 293T cells. Expression of proteins was determined with anti-AIRE and anti-α-tubulin antibodies by western blotting (WB). ( C ) Only the WT AIRE protein induces splicing of the KRT14 pre-mRNA. Relative splicing efficiency was determined and is presented for WT AIRE (black bar) and mutant AIREfsx (hashed bar) proteins as fold activation relative to the empty plasmid vector (C, white bar). Error bars represent SEM. ( D ) WT AIRE protein is expressed in 293T cells treated with FP. Expression of proteins was determined with anti-AIRE and anti-α-tubulin antibodies by western blotting (WB). ( E ) FP blocks AIRE-induced enhancement of KRT14 pre-mRNA splicing. Relative splicing efficiency was determined and is presented for the WT AIRE protein (black bars) as fold activation relative to the empty plasmid vector (C, white bars) for treated and untreated cells (FP, DMSO). Error bars represent SEM. ( F ) P-TEFb is enriched on the KRT14 gene in cells expressing the WT AIRE protein. ChIPs were performed with anti-CDK9 (αCDK9) and control IgG antibodies with lysates from 293T cells expressing the empty plasmid vector (C), WT AIRE or mutant AIREfsx proteins. Enrichment of specific KRT14 amplicons is presented as fold enrichment over IgG control (CDK9/IgG). White, black and striped bars represent values for the empty plasmid vector (C), WT AIRE and mutant AIREfsx proteins, respectively, with SEM depicted by error bars. The KRT14 gene and ChIP amplicons are depicted above the bar graph (promoter, Pr; 5′ of coding region, 5′; 3′ of coding region, 3′; polyadenylation signal, pA). ( G ) ChIP with anti-S2P (αS2P) antibodies. Expression levels for AIRE and α-tubulin proteins are presented below the bar graph (WB). Lysates from transfected cells were analyzed with specific antibodies by western blotting.

Article Snippet: Antibodies used for western blotting, co-immunoprecipitation (co-IP), ChIP or immunofluorescence: anti-AIRE (sc-33188 Santa Cruz Biotechnology), anti-Flag (M2 Sigma-Aldrich), anti-CDK9 (sc-484 Santa Cruz Biotechnology, ab10874 Abcam), anti-RNAPII (ab5408 Abcam, MMS-126 R Covance), anti-S2P (ab5095 Abcam), anti-GAL (sc-510 Santa Cruz Biotechnology), anti-GAPDH (AM4300 Ambion), anti-α Tubulin (DM1A Sigma-Aldrich), anti-U5-116 kD (A300-957A Bethyl labs) rabbit and mouse control IgG (Santa Cruz Biotechnology).

Techniques: Quantitative RT-PCR, Mutagenesis, Expressing, Western Blot, Activation Assay, Plasmid Preparation, Transfection

Journal: bioRxiv

Article Title: Inhibition of SF3B1 affects recruitment of P-TEFb to chromatin through multiple mechanisms

doi: 10.1101/2024.06.26.600844

Figure Lengend Snippet: ( A ) RT-PCR with primers amplifying the intron located between exons 4 and 5 of BRD2 or exons 2 and 3 of DNAJB1. HeLa cells were treated with DMSO or 1 µM PlaB for 30 min to 4 h. The location of the spliced and unspliced RNA is shown on the left of the panel. The percentages of unspliced RNA compared to total (spliced + unspliced) are shown below. ( B ) Representative images of immunofluorescence analysis of 5’EU incorporation in HeLa cells treated with DMSO, 100 µM DRB, a CDK9 inhibitor, or 1 µM PlaB for 1h. EU (green), DAPI (blue), scale bars: 50 µm. ( C ) Quantification of 5’EU intensity per nucleus for DMSO (blue), DRB (green), and PlaB (red). Boxplot settings are: min to max values with the box showing 25-75 percentile range. 394 nuclei were quantified per condition. Statistical test: Kruskal-Wallis test. P-value: **** < 0.0001. ( D ) Representative whole cell western blots of total RNAPII, RNAPII Ser2 phosphorylation, and β-actin (loading control) from HeLa cells treated with DMSO, 100 µM DRB for 4 h, or 1 µM PlaB for 30 min to 4h. ( E ) Quantification of the western blots shown in (D) presented as RNAPII Ser2P / total RNAPII following normalisation to the loading control and to the DMSO condition. Statistical test: Kruskal-Wallis test, n = 3 biological replicates. P-value: * < 0.05, ** < 0.01.

Article Snippet: Western blots were performed with NuPAGE Novex 4–12% Bis–Tris Protein Gels (Life Technologies) with the following primary antibodies: Rpb1 NTD (D8L4Y) Rabbit mAb (14958S, Cell Signaling Technology), Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb (13499S, Cell Signaling Technology), Rabbit anti-SF3b155/SAP155 (A300-996A, Bethyl Laboratories), Rabbit anti-Phospho-SF3B1 Ser129 (PD043, MBL International), Rabbit anti-Phospho-SF3B1 Thr142 (( )), Rabbit anti-Phospho-SF3B1 Thr211 (PA5-105427, Invitrogen), Rabbit anti-Phospho-SF3B1 Thr313 (D8D8V, Cell Signaling), Rabbit anti-Tat-SF1 (A302-023A, Bethyl Laboratories), Rabbit anti-CDK9 (ab76320, Abcam), Rabbit anti-Phospho-CDK9 (pThr186) (2549, Cell Signaling), Rabbit anti-Cyclin T1 (ab184703, Abcam), Rabbit anti-SNW1 (A300-784A, Bethyl Laboratories), Rabbit anti-c-MYC (sc-764, Santa Cruz Biotechnology), Rabbit anti-SC35 (SRSF2) (ab204916, Abcam), Rabbit anti-BRD4 (A301-985A100, Bethyl Laboratories), Rabbit anti-MCEF (AFF4) (A302-539A, Bethyl Laboratories), Mouse anti-U1A (3F9-1F7, Novus Biologicals), Rabbit anti-Actin (C-11) (sc-1615, Santa Cruz Biotechnology), Rabbit anti-α-Tubulin (2144S, Cell Signaling), Rabbit anti-Nucleolin (ab305947), Abcam), and Histone H3 (ab1791, Abcam).

Techniques: Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Western Blot, Control

Journal: bioRxiv

Article Title: Inhibition of SF3B1 affects recruitment of P-TEFb to chromatin through multiple mechanisms

doi: 10.1101/2024.06.26.600844

Figure Lengend Snippet: ( A ) Representative western blots of SF3B1, CDK9, CDK9 T186 phosphorylation, Cyclin T1, β-tubulin (loading control), and histone H3 (loading control) from chromatin, nucleoplasm, and cytoplasm fractions of HeLa cells treated with DMSO, 100 µM DRB, and 1 µM PlaB for 30 min. ( B ) Quantification of western blots of SF3B1, CDK9, and Cyclin T1 from chromatin, nucleoplasm, and cytoplasm fractions of HeLa cells following treatment with 1 µM PlaB for 30 min. The quantifications have been normalised to loading control and to DMSO, n = 4-5 biological replicates (including PlaB 30 min from Supplementary Figure 5C and PlaB from Supplementary Figure 6D). ( C ) Representative western blots of SF3B1, CDK9, Cyclin T1, U1-A (loading control), β-tubulin (loading control), and histone H3 (loading control) from chromatin, nucleoplasm, and cytoplasm fractions of HeLa cells treated with DMSO, ethanol, 0.02 µM Leptomycin B, or 1 µM PlaB. Treatments were performed as follow: Ethanol 1h/DMSO 30 min (-/-), LMB 1h/DMSO 30 min (+/-), Ethanol 1h/PlaB 30 min (-/+), LMB 1h/PlaB 30 min (+/+). ( D ) Total RNAPII ChIP-qPCR across the KPNB1 gene in HeLa cells treated with DMSO, ethanol, 0.02 µM Leptomycin B, or 1 µM PlaB as described in (C), n = 3 biological replicates. ( E ) Representative images of immunofluorescence analysis of 5’EU incorporation in HeLa cells treated with DMSO, ethanol, 100 µM DRB, 0.02 µM Leptomycin B, or 1 µM PlaB as described in (C). EU (green), DAPI (blue), scale bars: 50 µm. ( F ) Quantification of 5’EU intensity per nucleus for DMSO (blue), DRB (green), LMB (orange), PlaB (red), and LMB & PlaB (purple). Boxplot settings are: min to max values with the box showing 25-75 percentile range. 1,620 nuclei were quantified per condition. Statistical test: Kruskal-Wallis test. P-value: **** < 0.0001.

Article Snippet: Western blots were performed with NuPAGE Novex 4–12% Bis–Tris Protein Gels (Life Technologies) with the following primary antibodies: Rpb1 NTD (D8L4Y) Rabbit mAb (14958S, Cell Signaling Technology), Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb (13499S, Cell Signaling Technology), Rabbit anti-SF3b155/SAP155 (A300-996A, Bethyl Laboratories), Rabbit anti-Phospho-SF3B1 Ser129 (PD043, MBL International), Rabbit anti-Phospho-SF3B1 Thr142 (( )), Rabbit anti-Phospho-SF3B1 Thr211 (PA5-105427, Invitrogen), Rabbit anti-Phospho-SF3B1 Thr313 (D8D8V, Cell Signaling), Rabbit anti-Tat-SF1 (A302-023A, Bethyl Laboratories), Rabbit anti-CDK9 (ab76320, Abcam), Rabbit anti-Phospho-CDK9 (pThr186) (2549, Cell Signaling), Rabbit anti-Cyclin T1 (ab184703, Abcam), Rabbit anti-SNW1 (A300-784A, Bethyl Laboratories), Rabbit anti-c-MYC (sc-764, Santa Cruz Biotechnology), Rabbit anti-SC35 (SRSF2) (ab204916, Abcam), Rabbit anti-BRD4 (A301-985A100, Bethyl Laboratories), Rabbit anti-MCEF (AFF4) (A302-539A, Bethyl Laboratories), Mouse anti-U1A (3F9-1F7, Novus Biologicals), Rabbit anti-Actin (C-11) (sc-1615, Santa Cruz Biotechnology), Rabbit anti-α-Tubulin (2144S, Cell Signaling), Rabbit anti-Nucleolin (ab305947), Abcam), and Histone H3 (ab1791, Abcam).

Techniques: Western Blot, Control, Immunofluorescence

Journal: bioRxiv

Article Title: Inhibition of SF3B1 affects recruitment of P-TEFb to chromatin through multiple mechanisms

doi: 10.1101/2024.06.26.600844

Figure Lengend Snippet: ( A ) Co-immunoprecipitation of SF3B1 from whole cell extract of untreated HeLa cells followed by western blots of SF3B1, CDK9, Cyclin T1, and β-actin (negative control). ( B )Co-immunoprecipitation of Cyclin T1 from whole cell extract of untreated HeLa cells followed by western blots of SF3B1, CDK9, Cyclin T1, and β-actin (negative control). *: IgG. ( C ) Co-immunoprecipitation of SF3B1 from chromatin of HeLa cells treated with DMSO or 1 µM PlaB for 30 min followed by western blots of total RNAPII, SF3B1, CDK9, Cyclin T1, HTATSF1, SNW1, PRPF19, and histone H3. ( D ) Co-immunoprecipitation of Cyclin T1 from chromatin of HeLa cells treated with DMSO or 1 µM PlaB for 30 min followed by western blots of SF3B1, CDK9, Cyclin T1, HTATSF1, SNW1, β-actin, and histone H3. ( E ) Co-immunoprecipitation of SF3B1 from nucleoplasm of HeLa cells treated with DMSO or 1 µM PlaB for 30 min followed by western blots of total RNAPII, SF3B1, CDK9, Cyclin T1, HTATSF1, SNW1, PRPF19, and Nucleolin. ( F ) Co-immunoprecipitation of Cyclin T1 from nucleoplasm of HeLa cells treated with DMSO or 1 µM PlaB for 30 min followed by western blots of SF3B1, CDK9, Cyclin T1, HTATSF1, SNW1, and Nucleolin.

Article Snippet: Western blots were performed with NuPAGE Novex 4–12% Bis–Tris Protein Gels (Life Technologies) with the following primary antibodies: Rpb1 NTD (D8L4Y) Rabbit mAb (14958S, Cell Signaling Technology), Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb (13499S, Cell Signaling Technology), Rabbit anti-SF3b155/SAP155 (A300-996A, Bethyl Laboratories), Rabbit anti-Phospho-SF3B1 Ser129 (PD043, MBL International), Rabbit anti-Phospho-SF3B1 Thr142 (( )), Rabbit anti-Phospho-SF3B1 Thr211 (PA5-105427, Invitrogen), Rabbit anti-Phospho-SF3B1 Thr313 (D8D8V, Cell Signaling), Rabbit anti-Tat-SF1 (A302-023A, Bethyl Laboratories), Rabbit anti-CDK9 (ab76320, Abcam), Rabbit anti-Phospho-CDK9 (pThr186) (2549, Cell Signaling), Rabbit anti-Cyclin T1 (ab184703, Abcam), Rabbit anti-SNW1 (A300-784A, Bethyl Laboratories), Rabbit anti-c-MYC (sc-764, Santa Cruz Biotechnology), Rabbit anti-SC35 (SRSF2) (ab204916, Abcam), Rabbit anti-BRD4 (A301-985A100, Bethyl Laboratories), Rabbit anti-MCEF (AFF4) (A302-539A, Bethyl Laboratories), Mouse anti-U1A (3F9-1F7, Novus Biologicals), Rabbit anti-Actin (C-11) (sc-1615, Santa Cruz Biotechnology), Rabbit anti-α-Tubulin (2144S, Cell Signaling), Rabbit anti-Nucleolin (ab305947), Abcam), and Histone H3 (ab1791, Abcam).

Techniques: Immunoprecipitation, Western Blot, Negative Control

Journal: bioRxiv

Article Title: Inhibition of SF3B1 affects recruitment of P-TEFb to chromatin through multiple mechanisms

doi: 10.1101/2024.06.26.600844

Figure Lengend Snippet: ( A ) Western blots of total SF3B1, SF3B1 S129, T142, T211, and T313 phosphorylation, and histone H3 (loading control) from chromatin and nucleoplasm fractions of untreated HeLa cells. ( B ) Representative western blots of total SF3B1, SF3B1 S129, T142, T211, and T313 phosphorylation, and β-actin (loading control) from whole cell extract of HEK293 CDK9 analogue sensitive cells treated with DMSO, 1 µM PlaB or 15 µM 1-NA-PP1 for 30 min. ( C ) Quantification of western blots of total SF3B1 and SF3B1 S129, T142, T211, and T313 phosphorylation from whole cell extract of HEK293 CDK9 analogue sensitive cells treated with 1 µM PlaB or 15 µM 1-NA-PP1 for 30 min. The quantifications have been normalised to loading control and to DMSO. Statistical test: Kruskal-Wallis test, n = 2 - 3 biological replicates. P-value: * < 0.05. ( D ) Western blots of total SF3B1, SF3B1 S129, T142, T211, and T313 phosphorylation, histone H3 (loading control), and nucleolin (loading control) from chromatin and nucleoplasm fractions of HeLa cells treated with DMSO, 100 µM DRB for 4 h, or with 1 µM PlaB for 30 min to 4h. ( E ) Western blots of total SF3B1, SF3B1 S129, T142, T211, and T313 phosphorylation, and β-actin (loading control) from whole cell extract of HEK293 CDK12 analogue sensitive cells treated with DMSO or 7.5 µM 1-NM-PP1 for 30 min. Two biological replicates shown. ( F ) Quantification of western blots of total SF3B1 and SF3B1 S129, T142, T211, and T313 phosphorylation from whole cell extract of HEK293 CDK12 analogue sensitive cells treated with 7.5 µM 1-NM-PP1 for 30 min. The quantifications have been normalised to loading control and to DMSO. Statistical test: Kruskal-Wallis test, n = 4 biological replicates. P-value: * < 0.05.

Article Snippet: Western blots were performed with NuPAGE Novex 4–12% Bis–Tris Protein Gels (Life Technologies) with the following primary antibodies: Rpb1 NTD (D8L4Y) Rabbit mAb (14958S, Cell Signaling Technology), Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb (13499S, Cell Signaling Technology), Rabbit anti-SF3b155/SAP155 (A300-996A, Bethyl Laboratories), Rabbit anti-Phospho-SF3B1 Ser129 (PD043, MBL International), Rabbit anti-Phospho-SF3B1 Thr142 (( )), Rabbit anti-Phospho-SF3B1 Thr211 (PA5-105427, Invitrogen), Rabbit anti-Phospho-SF3B1 Thr313 (D8D8V, Cell Signaling), Rabbit anti-Tat-SF1 (A302-023A, Bethyl Laboratories), Rabbit anti-CDK9 (ab76320, Abcam), Rabbit anti-Phospho-CDK9 (pThr186) (2549, Cell Signaling), Rabbit anti-Cyclin T1 (ab184703, Abcam), Rabbit anti-SNW1 (A300-784A, Bethyl Laboratories), Rabbit anti-c-MYC (sc-764, Santa Cruz Biotechnology), Rabbit anti-SC35 (SRSF2) (ab204916, Abcam), Rabbit anti-BRD4 (A301-985A100, Bethyl Laboratories), Rabbit anti-MCEF (AFF4) (A302-539A, Bethyl Laboratories), Mouse anti-U1A (3F9-1F7, Novus Biologicals), Rabbit anti-Actin (C-11) (sc-1615, Santa Cruz Biotechnology), Rabbit anti-α-Tubulin (2144S, Cell Signaling), Rabbit anti-Nucleolin (ab305947), Abcam), and Histone H3 (ab1791, Abcam).

Techniques: Western Blot, Control

Journal: Molecular cell

Article Title: Mutant p53 Gains Its Function via c-Myc Activation upon CDK4 Phosphorylation at Serine 249 and Consequent PIN1 Binding

doi: 10.1016/j.molcel.2017.11.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-p53 (DO-1) Santa Cruz Biotechnology Cat# sc-126 AB_628082 Mouse monoclonal anti-p53 (DO-1) Santa Cruz Biotechnology Cat# sc-126 X Rabbit polyclonal anti-p53 (FL-393) Santa Cruz Biotechnology Cat# sc-6243 AB_653753 Normal mouse IgG Santa Cruz Biotechnology Cat# sc-2025 AB_737182 Rabbit monoclonal anti-GAPDH Cell Signaling Technology Cat# 5174 AB_10622025 Rabbit monoclonal anti-Histone H3 Cell Signaling Technology Cat# 4499 AB_10544537 Mouse monoclonal anti-cdc2 (CDK1) Cell Signaling Technology Cat# 9116P AB_2074795 Rabbit monoclonal anti-CDK2 Cell Signaling Technology Cat# 2546P AB_2276129 Rabbit monoclonal anti-CDK4 Cell Signaling Technology Cat# 12790P AB_2631166 Rabbit monoclonal anti-CDK6 Cell Signaling Technology Cat# 13331P Mouse monoclonal anti-CDK7 Cell Signaling Technology Cat# 2916P AB_10827986 Rabbit monoclonal anti-CDK9 Cell Signaling Technology Cat# 2316P AB_2291505 Mouse monoclonal anti-CDK4 Santa Cruz Biotechnology Cat# sc-56277 AB_1121419 Rabbit polyclonal anti-Phosphorylation of S249 This paper N/A Mouse monoclonal anti-Flag M2 Sigma-Aldrich Cat# F3165 AB_259529 Mouse monoclonal anti-Cyclin D1 Santa Cruz Biotechnology Cat# sc-20044 AB_627346 Mouse monoclonal anti-Cyclin D3 Cell Signaling Technology Cat# 2936P AB_10841292 Rabbit polyclonal anti-Pin1 Cell Signaling Technology Cat# 3722S AB_10692654 Rabbit polyclonal anti-Pin1 Santa Cruz Biotechnology Cat# sc-15340 AB_2237080 Mouse monoclonal anti-Pin1 Santa Cruz Biotechnology Cat# sc-46660 AB_628132 Rabbit monoclonal anti-PARP Cell Signaling Technology Cat# 9532S AB_10695538 Rabbit monoclonal anti-c-Myc Abcam Cat# ab32072 AB_731658 Mouse monoclonal anti-GFP Santa Cruz Biotechnology Cat# sc-9996 AB_627695 Mouse monoclonal anti-P21 WAF1 NeoMarkers Cat#MS-891-P1 Mouse monoclonal anti-beta-Actin Sigma-Aldrich Cat# A1978 AB_476692 Mouse monoclonal anti-beta-Actin Santa Cruz Biotechnology Cat# sc-47778 AB_626632 Mouse monoclonal anti-alpha-tubulin Sigma-Aldrich Cat# 00020911 AB_10013740 Peroxidase AffiniPure Goat Anti-Mouse IgG, Light Chain Specific for Western blotting after IP Jackson ImmunoResearch Cat# 115-035-174 AB_2338512 Peroxidase IgG Fraction Monoclonal Mouse Anti-Rabbit IgG, Light Chain Specific Jackson ImmunoResearch 211-032-171 AB_2339149 Mouse IgG Isotype Control Thermo Fisher Cat# 31903 AB_10959891 Rabbit IgG Isotype Control Thermo Fisher Cat# 31235 AB_243593 Bacterial and Virus Strains DH5a competent cells NEW ENGLAND BioLabs Cat# C2987H BL21(DE3) competent cells NEW ENGLAND BioLabs Cat# C2527H Stbl3 competent cells Thermo Fisher Cat# A10469 Biological Samples Hepatocellular carcinoma (HCC) tissue samples The First Affiliated Hospital of Nanchang University, Jiangxi, China.

Techniques: Western Blot, Control, Virus, Recombinant, CCK-8 Assay, Plasmid Preparation, Software