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: 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: Nucleic Acids Research

Article Title: Acute hypoxia affects P-TEFb through HDAC3 and HEXIM1-dependent mechanism to promote gene-specific transcriptional repression

doi: 10.1093/nar/gku611

Figure Lengend Snippet: Formation of an inactive P-TEFb complex in response to hypoxia. ( A ) Association of endogenous Cdk9 with HEXIM1 and Cyclin T1 in cells treated with normoxia (N) and hypoxia (H) in the presence or absence of IL-1β for 1 h. Whole cell lysates were immunoprecipitated (IP) with a rabbit anti-Cdk9 antibody followed by western blotting with goat anti-Cyclin T1 and a sheep anti-HEXIM1 antibody. Western blot with a monoclonal anti-Cdk9 antibody served as a control of total immunoprecipitated protein. To disrupt 7SK snRNA in ribonucleoprotein complexes, one set of cell lysates was treated with RNase A for 2 h prior to immunoprecipitation. Columns represent mean of densitometric quantification of 6 western blots obtained from independent immunoprecipitations ( n = 6); bars, SE; NT, without IL-1β treatment. * P < 0.05 as compared with normoxia; # P < 0.05 as compared with IL-1β-treated normoxic samples. ( B ) Association of cMyc/His-taged Cdk9 with HEXIM1 under normoxia (N) and hypoxia (H). Samples were immunoprecipitated using a monoclonal anti-cMyc antibody and analyzed as in (A). Western blot with a rabbit anti-Cdk9 antibody served as a control of total immunoprecipitated protein. Data shown represent one of two independent experiments. ( C ) Interactions of Cdk9-HEXIM1 and Cyclin T1-HEXIM1 were analyzed by P-LISA assay using rabbit anti-HEXIM1, mouse anti-Cdk9 and goat anti-Cyclin T1 antibodies. HeLa cells were exposured to hypoxia in the presence or absence of IL-1β for 1 h. PLA-signals of protein–protein interactions are shown as white speckles. Nuclei stained with SYTO 16 Nucleic Acid Stain in blue.

Article Snippet: The following antibodies were used in this study: anti-RNA Polymerase II (H-224), Fcp1 (H-300), Cdk9 (H-169) rabbit polyclonal, Cdk9 (D-7) mouse monoclonal, cyclin T1 (T-18), c-Myc (9E10) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Immunoprecipitation, Western Blot, Control, Protein-Protein interactions, Staining

Journal: Nucleic Acids Research

Article Title: Acute hypoxia affects P-TEFb through HDAC3 and HEXIM1-dependent mechanism to promote gene-specific transcriptional repression

doi: 10.1093/nar/gku611

Figure Lengend Snippet: The role of HDACs in hypoxic deacetylation of Cdk9 and Cyclin T1. HeLa cells were exposed for 1 h under normoxia or hypoxia 48 h after transfection with siRNA directed against HDAC1, HDAC2 or HDAC3. ( A ) Acetylated Cdk9 was analyzed by P-LISA assay using rabbit anti-AcLys and mouse anti-Cdk9 antibodies. ( B ) Acetylated Cyclin T1 was analyzed by P-LISA assay using rabbit anti-AcLys and goat anti-Cyclin T1 antibodies. P-LISA signals are shown in white and the nuclei in blue.

Article Snippet: The following antibodies were used in this study: anti-RNA Polymerase II (H-224), Fcp1 (H-300), Cdk9 (H-169) rabbit polyclonal, Cdk9 (D-7) mouse monoclonal, cyclin T1 (T-18), c-Myc (9E10) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Transfection

Journal: Nucleic Acids Research

Article Title: Acute hypoxia affects P-TEFb through HDAC3 and HEXIM1-dependent mechanism to promote gene-specific transcriptional repression

doi: 10.1093/nar/gku611

Figure Lengend Snippet: Hypoxic repression of Cdk9 and Cyclin T1 acetylation. ( A ) Intracellular distribution of Cdk9 acetylated at lysine residues in response to hypoxia. HeLa cells were pretreated with or without 500 nM TSA for 1 h and subsequently exposed to hypoxia for 1 h. Acetylated Cdk9 was analyzed by P-LISA assay using rabbit anti-AcLys and mouse anti-Cdk9 antibodies. ( B ) Acetylation of Cdk9 in response to 1 h exposure under normoxic (N) or hypoxic (H) gas mixtures with or without IL-1β. Immunoprecipitated Cdk9 was examined by western blotting with antibodies against acetyl-Lysin (AcLys) or Cdk9. All buffers used in this assay were supplemented with 100 ng/ml TSA to block endogenous HDAC activity. ( C ) Intracellular distribution of acetylated Cyclin T1 was analyzed by P-LISA assay using rabbit anti-AcLys and goat anti-Cyclin T1 antibodies. HeLa cells were pretreated with or without 500 nM TSA for 1 h followed by exposure to hypoxia for 1 h. Nucleic acids were stained using TOPRO-3 (blue).

Article Snippet: The following antibodies were used in this study: anti-RNA Polymerase II (H-224), Fcp1 (H-300), Cdk9 (H-169) rabbit polyclonal, Cdk9 (D-7) mouse monoclonal, cyclin T1 (T-18), c-Myc (9E10) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Immunoprecipitation, Western Blot, Blocking Assay, Activity Assay, Staining

Journal: Nucleic Acids Research

Article Title: Acute hypoxia affects P-TEFb through HDAC3 and HEXIM1-dependent mechanism to promote gene-specific transcriptional repression

doi: 10.1093/nar/gku611

Figure Lengend Snippet: Effect of depletion of class I HDACs on 7SK snRNA and cellular distribution of N-CoR/HDAC3 and Cdk9 in normoxic and hypoxic HeLa cells. ( A ) Effect of disruption of class I HDACs on the formation of an inactive endogenous P-TEFb complex in response to hypoxia. HeLa cells were exposed for 1 h to a normoxic (N) or hypoxic (H) gas mixture 48 h after transfection with siRNA. Whole cell lysates were immunoprecipitated with a goat anti-Cyclin T1 antibody and analyzed by western blot using rabbit anti-HEXIM1 and an anti-Cdk9 antibody. Direct western blots of HDAC1, HDAC2 and HDAC3 served as a control for siRNA-mediated knockdown. Western blot with an anti-Cyclin T1 antibody served as a control of total immunoprecipitated protein. ( B ) Localization of endogenous Cdk9 (red) and HDAC3 (green). Cells were treated with hypoxia, IL-1β or their combination for 2 h and analyzed by immunofluorescence staining. ( C ) Immunofluorescence showing cellular localization of endogenous HDAC3 (green) and N-CoR (red) in response to 2 h exposure to IL-1β alone or in combination with hypoxia. Nucleic acids were stained using TO-PRO-3 (blue).

Article Snippet: The following antibodies were used in this study: anti-RNA Polymerase II (H-224), Fcp1 (H-300), Cdk9 (H-169) rabbit polyclonal, Cdk9 (D-7) mouse monoclonal, cyclin T1 (T-18), c-Myc (9E10) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Disruption, Transfection, Immunoprecipitation, Western Blot, Control, Knockdown, Immunofluorescence, Staining

Journal: Nucleic Acids Research

Article Title: Acute hypoxia affects P-TEFb through HDAC3 and HEXIM1-dependent mechanism to promote gene-specific transcriptional repression

doi: 10.1093/nar/gku611

Figure Lengend Snippet: Hypoxia represses MCP-1 mRNA synthesis by inhibiting IL-1β-induced Ser2 phosphorylation of Pol II CTD. ( A ) Effect of hypoxia on MCP-1 and IL-8 mRNAs expressions. HeLa cells were exposed to normoxia or hypoxia without (NT) or with IL-1β for 1 h. mRNA levels were quantitated by Q-PCR in triplicate and normalized by 18S rRNA levels. Data are expressed as a percentage of expression under normoxia. * P < 0.05; ** P < 0.01, as compared with normoxia; # P < 0.05; ## P < 0.01, as compared with IL-1β-treated normoxic samples. ( B ) Pol II Rbp1 promoter occupancy of the IL-8 and MCP-1 genes analyzed by ChIP assay after treatment with IL-1β and/or hypoxia for the indicated periods of time. ( C ) The phosphorylation status of Ser5 and Ser2 residues of Pol II CTD set on the MCP-1 and IL-8 gene promoters in the cells treated with IL-1β under normoxia or hypoxia for the indicated time periods. ( B-C ) Immunoprecipitated DNA was quantified by Q-PCR in triplicate and after normalization by 10% input expressed as a percentage of binding at a 0 min time point. * P < 0.05; ** P < 0.01, as compared with binding at 0 min. ( D ) ChIP assay analysis of P-TEFb enrichment on the MCP-1 and IL-8 promoters. HeLa cells were treated with IL-1β and/or hypoxia for the indicated periods of time. DNA was immunoprecipitated using Cdk9 and Cyclin T1 antibodies and quantified by Q-PCR Data expressed as a percentage of binding in normoxic cells at 30 min or 60 min time point after normalization by 10% input. ** P <0.01 as compared with normoxia; ## P < 0.01, as compared with IL-1β-treated normoxic samples.

Article Snippet: The following antibodies were used in this study: anti-RNA Polymerase II (H-224), Fcp1 (H-300), Cdk9 (H-169) rabbit polyclonal, Cdk9 (D-7) mouse monoclonal, cyclin T1 (T-18), c-Myc (9E10) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, Expressing, Immunoprecipitation, Binding Assay

Journal: Nucleic Acids Research

Article Title: Acute hypoxia affects P-TEFb through HDAC3 and HEXIM1-dependent mechanism to promote gene-specific transcriptional repression

doi: 10.1093/nar/gku611

Figure Lengend Snippet: Microarray analysis of primary responses to hypoxia and identification of HEXIM1-dependent genes. HeLa cells were transfected with control (siScr) and HEXIM1-targeted (siHEXIM) double-strand siRNA and 48 h after treated with IL-1β in combination with normoxia (N+IL) or hypoxia (H+IL) for 1 h. ( A ) Venn diagram of genes down-regulated by hypoxia and their dependence on HEXIM1. ( B ) Venn diagram of genes up-regulated by hypoxia and their dependence on HEXIM1. ( C ) Microarray identification of HEXIM1-dependent genes among the probes with decreased expression in hypoxia. ( D ) Microarray identification of HEXIM1-dependent genes among the probes with increased expression in hypoxia. ( E ) The model of hypoxic inhibition of P-TEFb. Hypoxia enhances formation of an inactive P-TEFb complex with its endogenous inhibitors Hexim1 and 7SK snRNA. This process requires the HDAC3/N-CoR repressor complex. Inhibition of P-TEFb is mediated by HDAC-dependent deacetylation of P-TEFb subunits Cdk9 and Cyclin T1. HDAC2 is required for active P-TEFb via an unknown mechanism.

Article Snippet: The following antibodies were used in this study: anti-RNA Polymerase II (H-224), Fcp1 (H-300), Cdk9 (H-169) rabbit polyclonal, Cdk9 (D-7) mouse monoclonal, cyclin T1 (T-18), c-Myc (9E10) antibodies were purchased from Santa Cruz Biotechnology.

Techniques: Microarray, Transfection, Control, Expressing, Inhibition