Ha cdk9

Manufactured by Addgene

HA-CDK9 is a recombinant protein that contains the Hemagglutinin (HA) tag fused to the Cyclin-Dependent Kinase 9 (CDK9) protein. CDK9 is a subunit of the positive transcription elongation factor b (P-TEFb) complex, which plays a crucial role in the regulation of gene expression.

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Lab products found in correlation

Cisplatin, by J&K Scientific (1 mentions) Enhanced chemiluminescent plus reagents, by GE Healthcare (1 mentions) Turbofect transfection reagent, by Thermo Fisher Scientific (1 mentions) V5 turboid nes pcdna3, by Addgene (1 mentions) Anti hexim1, by Fortis Life Sciences (1 mentions) Anti pol 2, by Santa Cruz Biotechnology (1 mentions) Anti cyclin t1, by Santa Cruz Biotechnology (1 mentions) Anti actin, by Abcam (1 mentions) Anti nucleophosmin b23, by Santa Cruz Biotechnology (1 mentions) Anti acetyl lysine, by Cell Signaling Technology (1 mentions) Anti flag m2 agarose, by Merck Group (1 mentions) Anti cdk9, by Santa Cruz Biotechnology (1 mentions) Anti nucleolin, by Santa Cruz Biotechnology (1 mentions) Anti tubulin, by Merck Group (1 mentions) Anti p53, by Abcam (1 mentions) Anti flag, by Merck Group (1 mentions)

3 protocols using ha cdk9

Cytotoxic Metal Complexes Evaluation

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Compounds 21 and 22 (commercially available, purity >95%), and cisplatin were purchased from J&K Scientific Ltd., Hong Kong, China. Compound 20 was purchased from Medchemexpress (Shanghai, China), and complexes 1–19 were synthesized. The purity of all complexes were validated by combustion using automatic analyzers (Atlantic Microlab Inc., Norcross, GA, USA) to confirm >95% purity. All the complexes were dissolved in dimethyl sulfoxide (DMSO). Lipo3000, MTT kit were purchased from Sigma–Aldrich (Santa Clara, CA, USA). Plasmids c-Myc, Mcl-1, HA-CDK9 were purchased from Addgene. TurboFect™ Transfection Reagent was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Proteins bands were detected using enhanced chemiluminescent Plus reagents (GE Healthcare) and analyzed by Image Lab. Dulbecco's modified Eagle's medium (DMEM), Fetal bovine serum (FBS) were obtained from Gibco BRL (Gaithersburg, MD, USA). All other reagents and chemicals were obtained from commercial sources.

Cheng S.S., Qu Y.Q., Wu J., Yang G.J., Liu H., Wang W., Huang Q., Chen F., Li G., Wong C.Y., Wong V.K., Ma D.L, & Leung C.H. (2021). Inhibition of the CDK9–cyclin T1 protein–protein interaction as a new approach against triple-negative breast cancer. Acta Pharmaceutica Sinica. B, 12(3), 1390-1405.

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Construction of V5-TurboID-tagged CDK9 Plasmid

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To build the TurboID-bait assay, we first constructed a V5-TurboID-tagged CDK9 plasmid (pDL2518) through Gibson assembly method. The template plasmids HA-CDK9 (a gift from Andrew Rice, plasmid # 28102) and V5-TurboID-NES_pCDNA3 (a gift from Alice Ting, plasmid # 107169) were purchased from Addgene. The primer pair (5′-cagtctgcggtctgccgaaaagctgcagATGGCAAAGCAGTACGACTCGGTGGAGT-3′ and 5′-cagggtcaggcgctccaggggaggcagTCAGAAGACGCGCTCAAACTCCGTCTGGT-3′) and the template HA-CDK9 were used to clone the CDK9 fragment. The two primer pairs: pair-1 (5′- ACCAGACGGAGTTTGAGCGCGTCTTCTGActgcctcccctggagcgcctgaccctg-3′ and 5′-caatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgac-3′); pair-2 (5′-gtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattg-3′ and 5′-ACTCCACCGAGTCGTACTGCTTTGCCATctgcagcttttcggcagaccgcagactg-3′) and the template V5-TurboID-NES_pCDNA3 were used to clone the V5-TurboID fragment. The PCR fragments were assembled using NEBuilder^® HiFi DNA Assembly Master Mix (Cat. No. E2621, NEB, Ipswich, MA, USA). DNA sequences in all these plasmids were authenticated by automatic sequencing.

Pei J., Xiao Y., Liu X., Hu W., Sobh A., Yuan Y., Zhou S., Hua N., Mackintosh S.G., Zhang X., Basso K.B., Kamat M., Yang Q., Licht J.D., Zheng G., Zhou D, & Lv D. (2023). Piperlongumine (PL) conjugates induce targeted protein degradation. Cell chemical biology, 30(2), 203-213.e17.

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Molecular Tools for SIRT7 Research

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Plasmids encoding hSIRT7, sh-hSIRT7, Flag-hSIRT7 (1 (link)), Flag/HA-SIRT7 and clonal lines that stably express Flag/HA-SIRT7 (13 ) have been described. SIRT7 truncation mutants were generated by PCR and cloned into pCMV-Flag vector. Oligonucleotides used for plasmid construction and mutagenesis are listed in Supplementary Table S1. Expression vectors encoding HA-CDK9 and Flag-RPB1 were from Addgene. Antibodies against UBF, PAF53, RPA116 and SIRT7 have been described (13 ,17 (link),18 (link)). The following commercial antibodies were used: anti-acetyl lysine (Cell Signaling Technology, 9441), anti-actin (Abcam, ab8227), anti-CDK9 (Santa Cruz, sc-484 (C20)), anti-CHD4/Mi2 (Santa Cruz, sc-55606), anti-cyclin T1 (Santa Cruz, sc-10750), anti-DDX21 (Santa Cruz, sc-376953), anti-Flag (Sigma, F3165), anti-HEXIM1 (Bethyl Laboratories, A303-113A), anti-hnRNPK/J (Santa Cruz, sc-32307), anti-hnRNPU (Santa Cruz, sc-32315), anti-nucleolin (Santa Cruz, sc-13057), anti-nucleophosmin/B23 (Santa Cruz, sc-53175), anti-p53 (Abcam, ab31333), anti-Pol II (Santa Cruz, sc-56767 and sc-899 (N20)), anti-pSer2-Pol II (Active Motif, 6108), anti-pSer5-Pol II (Abcam, ab5408) and anti-tubulin (Sigma, clone B-5-1-2, T6074). anti-Flag M2 agarose was from Sigma (F1804). Secondary antibodies were from Dianova (111-035-144 and 115-035-062).

Blank M.F., Chen S., Poetz F., Schnölzer M., Voit R, & Grummt I. (2017). SIRT7-dependent deacetylation of CDK9 activates RNA polymerase II transcription. Nucleic Acids Research, 45(5), 2675-2686.

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