Cdk13

Manufactured by Fortis Life Sciences

CDK13 is a cyclin-dependent kinase enzyme that plays a role in the regulation of gene transcription. It is involved in the phosphorylation of the C-terminal domain of the RNA polymerase II complex, which is a key step in the initiation and elongation of transcription. CDK13 is expressed in various cell types and is important for cellular processes such as cell cycle regulation and gene expression.

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3 protocols using cdk13

Western Blot Analysis of CDK and RNA Polymerase II Phosphorylation

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Cells were lysed in Cell Signaling Lysis Buffer (Cell Signaling Technology) supplemented with Complete, EDTA free Protease Inhibitor Cocktail (Roche Diagnostics) and PhosSTOP Phosphatase Inhibitor (Roche Diagnostics). Protein concentrations were determined by the Bradford protein assay. Protein samples were separated by SDS-PAGE and transferred to PVDF membranes. Membranes were incubated with primary antibodies directed against CDK7 (Cell Signaling Technology Cat. No. 2090S), CDK12 (Cell Signaling Technology Cat. No. 11973), CDK13 (Bethyl Cat. No. A301-458A), Cyclin K (Abcam ab130475), p-Ser2-RNA-Pol II (Bethyl Cat. No. A300654A), p-Ser5-RNA-Pol II (Bethyl Cat. No. A300-655A), p-Ser7-RNA-Pol II (Millipore Cat. No. 04-1570), total RNA-Pol II (Santa Cruz Cat. No. sc-17798), PARP (Cell Signaling Technology Cat. No. 9542), RAD51 (Cell Signaling Technology Cat. No. 8875), FLI1 (Santa Cruz Biotechnology Cat. No. sc-356), Vinculin (Abcam Cat. No. ab18058), GAPDH (Santa Cruz Biotechnology Cat. No. sc-137179), and β-Actin (Cell Signaling Technology Cat. No. cs3700). Horseradish peroxidase (HRP) conjugated secondary antibodies were used. Blots were visualized by enhanced chemi-luminescence (ThermoFisher Scientific).

Iniguez A.B., Stolte B., Wang E.J., Conway A.S., Alexe G., Dharia N.V., Kwiatkowski N., Zhang T., Abraham B.J., Mora J., Kalev P., Leggett A., Chowdhury D., Benes C.H., Young R.A., Gray N.S, & Stegmaier K. (2018). EWS/FLI confers tumor cell synthetic lethality to CDK12 inhibition in Ewing sarcoma. Cancer cell, 33(2), 202-216.e6.

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Western Blot Analysis of Cullin Proteins

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PBS-washed cell pellets were lysed in 50mM Tris pH 7.9, 8M Urea and 1% CHAPS and incubated with shaking at 4ºC for at least 30min. 20μg of supernatants were run and transferred for detection. Antibodies used: CUL1 (1:1000, Santa Cruz Biotechnology, sc-1276), CUL2 (1:500, Sigma-Aldrich, SAB2501565-100), CUL3 (1:1000, Cell Signaling Technology, 2759), CUL4A (1:1000, Cell Signaling Technology, 2699S), CUL4B (1:1000, Proteintech, 12916-1-AP), CUL5 (1:1000, Santa Cruz Biotechnology, sc-373822), UBE2M (1:1000, Santa Cruz Biotechnology, sc-390064), DDB1 (1:1000, Cell Signaling Technology, 5428S), cyclin K (1:5000, Bethyl, A301-939A), CDK12 (1:1000, Cell Signaling Technology, 11973S), CDK13 (1:1000, Bethyl, A301-458A), RBM39 (1:500, Santa Cruz Biotechnology sc-376531), V5 (1:1000 Cell Signaling Technology, 13202), Ubiquityl-Histone H2A (K119) (1:1000, Cell Signaling, 8240-20), MLH1 (1:1000, Cell Signaling Technology, 3515T). ACTIN (1:10000, Sigma-Aldrich, A5441), VINCULIN (1:1000, Santa Cruz Biotechnology, sc-25336). Secondary antibodies anti-mouse/rabbit/goat (1:10000, Jackson ImmunoResearch 115-035-003, 111-035-003 and 705-035-003) and anti-rabbit Alexa Fluor 488 (1:1000, Invitrogen, A21441).

Mayor-Ruiz C., Bauer S., Brand M., Kozicka Z., Siklos M., Imrichova H., Kaltheuner I.H., Hahn E., Seiler K., Koren A., Petzold G., Fellner M., Bock C., Müller A.C., Zuber J., Geyer M., Thomä N.H., Kubicek S, & Winter G.E. (2020). Rational discovery of molecular glue degraders via scalable chemical profiling. Nature chemical biology, 16(11), 1199-1207.

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Antibody Characterization for RNA Polymerase II

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The following antibodies were used: RNAPII CTD S2 (Bethyl cat# A300-654A, 1:10,000); RNAPII CTD S5 (Bethyl cat# A300-655A, 1:10,000); RNAPII (Santa Cruz cat# sc-899, 1:1000); RNAPII CTD S7 (Millipore cat# 041570, 1:10,000) RNAPII CTD Thr4 (Active Motif cat# 61361, 1:1000) cleaved PARP (Cell Signaling cat# 9541, 1:1000); β-Actin (Cell Signaling cat# 4967, 1:4000); CDK12 (Cell Signaling cat# 11973 s, 1:1000); CDK13 (Bethyl cat# A301-458A, 1:1000); CDK13 antibody (1:1000) used for WB in Supplementary Fig. 2e was kindly provided by Dr. Arno Greenleaf; γ-H2AX (Cell Signaling cat# 9718, 1:200), RAD51 (GeneText cat# GTX70230, 1:1000), BRCA1 (Cell signaling cat# 9010S, 1:500), BARD1 (Santa Cruz Biotechnology cat# sc11438, 1:500), Alexa-488 (Molecular Probes cat#A11008, 1:300).

Krajewska M., Dries R., Grassetti A.V., Dust S., Gao Y., Huang H., Sharma B., Day D.S., Kwiatkowski N., Pomaville M., Dodd O., Chipumuro E., Zhang T., Greenleaf A.L., Yuan G.C., Gray N.S., Young R.A., Geyer M., Gerber S.A, & George R.E. (2019). CDK12 loss in cancer cells affects DNA damage response genes through premature cleavage and polyadenylation. Nature Communications, 10, 1757.

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