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Anti cdk9

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Anti-CDK9 is a laboratory equipment product designed for the detection and analysis of the cyclin-dependent kinase 9 (CDK9) protein. CDK9 is a key regulator of gene transcription and plays a crucial role in cellular processes. The Anti-CDK9 product provides researchers with a tool to study the expression and activity of CDK9 in various biological systems.

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Lab products found in correlation

Anti cyclin t1, by Santa Cruz Biotechnology (4 mentions) Anti flag, by Merck Group (3 mentions) Criterion gel, by Bio-Rad (2 mentions) Anti ankrd1, by Santa Cruz Biotechnology (2 mentions) Anti vdac1, by GenScript (2 mentions) Anti brd4, by Fortis Life Sciences (2 mentions) Anti p300, by Santa Cruz Biotechnology (2 mentions) Anti pol 2, by Santa Cruz Biotechnology (2 mentions) Anti hexim1, by Fortis Life Sciences (2 mentions) Anti gapdh, by Merck Group (2 mentions) Anti g3bp1, by Merck Group (1 mentions) Anti phosphoakt ser473 and anti akt, by Cell Signaling Technology (1 mentions) Anti actin, by Santa Cruz Biotechnology (1 mentions) Viia 7 system, by Thermo Fisher Scientific (1 mentions) Chip dna purification kit, by Active Motif (1 mentions) Anti igg, by Cell Signaling Technology (1 mentions) Mini protean tgx gel, by Bio-Rad (1 mentions) Odyssey clx imager, by LI COR (1 mentions) Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions) Trans blot turbo transfer system, by Bio-Rad (1 mentions)

15 protocols using anti cdk9

1

Subcellular Protein Fractionation and Analysis

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Cells lysate were fractionated into cellular compartments using a Subcellular ProteoExtract Kit (Calbiochem), according to the manufacturer’s protocol. The protein was analyzed on a 4% to 20% gradient SDS-PAGE (Criterion gels, Bio-Rad) and transferred to nitrocellulose membrane for western blotting. Anti-G3bp1 (Millipore), Anti-Myosin (slow, skeletal) (Sigma), anti-phosphoAkt (Ser473) and anti-Akt (Cell Signaling), anti-Gapdh (Millipore), anti-Cdk9 (Santa Cruz Biotech), anti-Gtf2b (Millipore), anti-Ankrd1 (Santa Cruz Biotech), anti-Vdac1 (Genscript), anti-H2b (Upstate), anti-Eif4e (Genscript) and anti-Actin (Santa Cruz Biotech)
He M., Yang Z., Abdellatif M, & Sayed D. (2015). GTPase Activating Protein (Sh3 Domain) Binding Protein 1 Regulates the Processing of MicroRNA-1 during Cardiac Hypertrophy. PLoS ONE, 10(12), e0145112.
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2

Chromatin Immunoprecipitation (ChIP) Assay Protocol

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Chromatin immunoprecipitation (ChIP) assays were performed according to manufacturer's protocols (Millipore, Bedford, MA, USA) with slight modifications. MDA-MB-231 cells treated with compound 1 or 15 (5 μM) or DMSO control for 12 h and cross-linked by incubating with 1% (vol/vol) formaldehyde-containing medium for 10 min at 37°C. Then chromatin with DNA fragments between 200 and 1000 base pairs. Anti-CDK9 (Santa Cruz Biotechnology) and Anti-IgG (Cell Signaling Technology) was used to capture DNA fragments. Purification was performed using the ChIP DNA Purification Kit (Active Motif, Carlsbad, CA, USA). ChIP-PCR analysis were performed by real-time qPCR (ViiA™ 7 System, Life Technologies). The PCR primers for the target promoters are shown in Table S1.
Cheng S., Yang G.J., Wang W., Ma D.L, & Leung C.H. (2021). Discovery of a tetrahydroisoquinoline-based CDK9-cyclin T1 protein–protein interaction inhibitor as an anti-proliferative and anti-migration agent against triple-negative breast cancer cells. Genes & Diseases, 9(6), 1674-1688.
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3

Protein Extraction and Western Blotting

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Protein was extracted from mouse tissues using Tissue Protein Extraction Reagent (T-PER) plus Halt Protease Inhibitor Cocktail (both from Thermo Scientific, Waltham, MA, USA), following manufacturer’s instructions. A total of 50 μg of protein per sample was run on 4%–20% Mini-PROTEAN TGX Gels and transferred using the Trans-Blot Turbo Transfer System and Nitrocellulose RTA Transfer Kit (all from Bio-Rad Laboratories, Hercules, CA, USA). Membranes were then immunoblotted with the antibodies listed in Table 1.

Antibodies Used for Western Blotting

AntibodyCompanyConcentration Used
Anti-CAB39Cell Signaling Technology, Danvers, MA, USA1:750
Anti-MIFR&D Systems, Minneapolis, MN, USA1:750
Anti-Cdk9Santa Cruz Biotechnology, Dallas, TX, USA1:1,000
Anti-dHandSanta Cruz Biotechnology, Dallas, TX, USA1:500
Anti-beta Tubulin Loading ControlLife Technologies, Carlsbad, CA, USA1:2,000
Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 680Life Technologies, Carlsbad, CA, USA1:5,000
Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 680Life Technologies, Carlsbad, CA, USA1:5,000
Blots were imaged on an Odyssey CLx Imager (LI-COR Biosciences, Lincoln, NE, USA). Quantification was performed using ImageJ 1.46r (NIH).
Course M.M., Gudsnuk K., Desai N., Chamberlain J.R, & Valdmanis P.N. (2019). Endogenous MicroRNA Competition as a Mechanism of shRNA-Induced Cardiotoxicity. Molecular Therapy. Nucleic Acids, 19, 572-580.
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4

Comprehensive Antibody Analysis Protocol

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The following antibodies were used for IP and/or western blot analysis: anti-β-actin (#4970; Cell Signalling), anti-CDK9 (sc-13130; Santa Cruz), anti-CNOT1 (14276-1-AP; Protein Tech), anti-CNOT7 (H00029883; Abnova), anti-FLAG (M2; Sigma), anti-hnRNPD (Q14103; Millipore), anti-HA (1867423; Roche), anti-IGF2BP1 (sc-21026; Santa Cruz), anti-KHSRP (ab83291; Abcam), anti-LARP7 (LaRP7-101AP; FabGennix Inc.), anti-LDLR (AF2148; BD Biosciences), anti-Myc (9E10; Roche), anti-phospho-ERK (#9101s; Cell Signalling), anti-phospho-MAPKAPK2 (#3007s; Cell Signalling), anti-phospho-RSK (sc-17033; Santa Cruz), anti-ZFP36L1 (#2119; Cell Signalling), anti-phospho-S6 (#2211; Cell Signalling).
Adachi S., Homoto M., Tanaka R., Hioki Y., Murakami H., Suga H., Matsumoto M., Nakayama K.I., Hatta T., Iemura S.I, & Natsume T. (2014). ZFP36L1 and ZFP36L2 control LDLR mRNA stability via the ERK–RSK pathway. Nucleic Acids Research, 42(15), 10037-10049.
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5

Immunoblotting Analysis of Adipogenic Factors

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Total cell proteins extracted in RIPA buffer and cell extracts were separated by 10% SDS-PAGE and subjected to immunoblotting as previously described35 . The primary antibodies used in this study were as follows: anti-PPARγ2 (#MAB3630, Chemicon, Temecula, CA); anti-BRD4, which was generated against the BRD4 C-terminal peptide (CFQSDLLSIFEENLF) by a custom antibody service (Sigma-Aldrich); anti-BRD2 (#5848, Cell Signalling Technology, Danvers, MA); anti-TRAP220 (#sc-8998, Santa Cruz Biotechnology, Dallas, TX); anti-CDK9 (#sc-8338, Santa Cruz Biotechnology); and anti-CYCLIN T1 (#ab2098, Abcam, Cambridge, UK). The ADIPOQ protein levels in medium and serum were measured by ELISA (Mouse Adiponectin ELISA Kit; CycLex, Nagano, Japan).
Sakurai N., Inamochi Y., Inoue T., Hariya N., Kawamura M., Yamada M., Dey A., Nishiyama A., Kubota T., Ozato K., Goda T, & Mochizuki K. (2017). BRD4 regulates adiponectin gene induction by recruiting the P-TEFb complex to the transcribed region of the gene. Scientific Reports, 7, 11962.
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6

Chromatin Immunoprecipitation Analysis of Androgen Receptor Signaling

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ChIP analysis was as previously described (25 (link)). Antibodies used were anti- AR (Santa Cruz, Cat. sc-816), anti-pAR-S81 (EMD Millipore, Cat. 07–1375), anti-pRNA Pol II Ser2 (Abcam, Cat., ab5095), anti-pRNA Pol II Ser5 (Abcam, Cat. ab5131), anti-CDK9 (Santa Cruz, Cat. sc-8338), anti-cyclin T1 (Santa Cruz, Cat. sc-10750), anti-BRD4 (Bethyl, Cat. A301-985A); anti-p300 (Santa Cruz, Cat. sc-585), anti-H3K27Ac (Abcam, Cat. ab4729), anti-PP1α (combined Santa Cruz sc-6104 and sc-6105). Primers for ChIP were: PSA-enhancer: Forward, 5΄-GCCTGGATCTGAGAGAGATATCATC-3΄; Reverse, 5΄-ACACCTTTTTTTTTCTGGATTGTTG-3΄; PSA-promoter: Forward, 5΄-TCCTGAGTGCTGGTGTCTTAG-3΄; Reverse, 5΄-CAGGATGAAACAGAAACAGGG-3΄; KLK2-Enhancer: Forward, 5΄-GCCTTTGCTCAGAAGACACA-3΄; Reverse, 5΄-ACAAGAGTGGAAGGCTCTGG -3΄; KLK2-Promoter: Forward, 5΄-CCTGTTGCTGTTCATCCTGA-3΄; Reverse, 5΄-CCTATGGATCATGGAGATGTGA-3΄.
Liu X., Gao Y., Ye H., Gerrin S., Ma F., Wu Y., Zhang T., Russo J., Cai C., Yuan X., Liu J., Chen S, & Balk S.P. (2017). Positive feedback loop mediated by protein phosphatase 1α mobilization of P-TEFb and basal CDK1 drives androgen receptor in prostate cancer. Nucleic Acids Research, 45(7), 3738-3751.
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7

Western Blot Antibody Panel for Protein Expression

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Western blot was performed as described [58] (link), [59] (link). The primary antibodies used in this study were: anti-Npac (Catalog No. 14833-1-AP, Proteintech), anti-NP60 (Catalog No. sc-390601, Santa Cruz, Dallas, TX), anti-β-actin (Catalog No. sc-81178, Santa Cruz), anti-Oct4 (Catalog No. sc-8628, Santa Cruz), anti-Nanog (Catalog No. sc-33760, Santa Cruz), anti-Sox2 (Catalog No. sc-99000, Santa Cruz), anti-p-ERK (Catalog No. 4370, Cell Signaling Technology, Danvers, MA), anti-ERK (Catalog No. 137F5, Cell Signaling Technology), anti-histone H3K36me3 (Catalog No. ab9050, Abcam), anti-Pol II (Catalog No. sc-899, Santa Cruz), anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) (Catalog No. ab5095, Abcam), anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) (Catalog No. ab140509, Abcam), anti-Cyclin T1 (Catalog No. sc-10750, Santa Cruz), anti-Cdk9 (Catalog No. sc-484, Santa Cruz), anti-mouse IgG (Catalog No. sc-2025, Santa Cruz), anti-goat IgG (Catalog No. sc-2028, Santa Cruz), and anti-rabbit IgG (Catalog No. sc-2027, Santa Cruz).
Yu S., Li J., Ji G., Ng Z.L., Siew J., Lo W.N., Ye Y., Chew Y.Y., Long Y.C., Zhang W., Guccione E., Loh Y.H., Jiang Z.H., Yang H, & Wu Q. (2021). Npac Is A Co-factor of Histone H3K36me3 and Regulates Transcriptional Elongation in Mouse Embryonic Stem Cells. Genomics, Proteomics & Bioinformatics, 20(1), 110-128.
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8

SDS-PAGE Protein Separation and Antibody Immunoblotting

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Ten microgram of protein samples, each, were subjected to electrophoresis on 4–15% SDS-PAGE (Criterion gels; Bio-Rad). The antibodies used for Western blot analysis included anti-ANKRD1 (Santa Cruz), anti-CDK9 (Santa Cruz), anti-Myosin, slow (Sigma), anti-TFIIB (Millipore), anti-GAPDH (Chemicon), anti-VDAC1 (Genscript), Anti-GFP (BD Biosciences), Anti-Luciferase (Novus Biologicals), anti-Nelf-A (SantaCruz). The signal was detected and quantified by the Odyssey Imaging System (LI-COR).
Sayed D., Yang Z., He M., Pfleger J.M, & Abdellatif M. (2014). Acute Targeting of General Transcription Factor IIB Restricts Cardiac Hypertrophy via Selective Inhibition of Gene Transcription. Circulation. Heart failure, 8(1), 138-148.
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9

Antibodies for ChIP and Western Blotting

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The following antibodies were used for chromatin immunoprecipitation (ChIP) or western blotting (WB): anti-PFDN5 (Santa Cruz, sc-271150, WB); anti-GAPDH (Santa Cruz, sc-47724, WB); anti-PFDN2 (Bethyl, A304-807A, WB); anti-Rpb1 CTD Ser2 phosphorylated (Abcam, ab5095, ChIP and WB); anti-Rpb1 CTD Ser5 phosphorylated (Abcam, ab5131, ChIP and WB); anti-Rpb1 8WG16 (Thermo Fisher MA1-10882, ChIP); anti-FLAG (Sigma, F3165, ChIP); anti-U2AF65 (Santa Cruz, sc-53942, ChIP and WB); anti-PRPF19 (Lührmann Laboratory, ChIP and WB); anti-PRGL1 (Lührmann Laboratory, WB); anti-CDK9 (Santa Cruz, sc-13130, ChIP and WB); anti-vinculin (Santa Cruz, sc-25336, WB).
Payán-Bravo L., Fontalva S., Peñate X., Cases I., Guerrero-Martínez J.A., Pareja-Sánchez Y., Odriozola-Gil Y., Lara E., Jimeno-González S., Suñé C., Muñoz-Centeno M.C., Reyes J.C, & Chávez S. (2021). Human prefoldin modulates co-transcriptional pre-mRNA splicing. Nucleic Acids Research, 49(11), 6267-6280.
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10

Preparation and Analysis of HEK293 Nuclear Extracts

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HEK293 nuclear extracts were prepared as described previously35 (link). For immunoprecipitations, extracts were incubated overnight with 3 μg of the desired antibody pre-bound to protein G-sepharose (Pierce). In some case protein extracts were treated with RNaseA (20 μg ml−1). Immunocomplexes were eluted in 2× Laemmli buffer and resolved in an 8% acrylamide gel. For western blots the following antibodies were used according to manufacturer instructions: anti-NOP58 (Bethyl A302-718A); anti-fibrillarin (Cell Signaling C13C3); anti-DKC1 (Gene Tex GTX109000); anti-Flag (Sigma); anti-DDX21 (Novus Biologicals NB100-1781); anti-LARP7 (a gift from D. H. Price); anti-CDK9 (Santa Cruz Biotechnology sc-484); anti-cyclinT1 (Santa Cruz Biotechnology sc-10750); and anti-HEXIM1 (Bethyl A303-113A). All antibodies have been previously validated unless otherwise specified.
Calo E., Flynn R.A., Martin L., Spitale R.C., Chang H.Y, & Wysocka J. (2014). RNA helicase DDX21 coordinates transcription and ribosomal RNA processing. Nature, 518(7538), 249-253.
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