Cyclin k

The following antibodies were used for immunoblots: Pol II CTD Ser-2 (cat# 04-1571, 1:1000), Ser-5 (cat# 04-1572, 1:5000), and Ser-7 (cat# 04-1570, 1:1000) phosphoantibodies (Millipore); Total Pol II (Santa Cruz cat# sc-899, 1:500); CDK7 (Santa Cruz cat# sc-723, 1:500); CDK9 (Bethyl cat# A303-493A, 1:1000); CDK12 and CDK13 (Arno Greenleaf, 1:1000); cyclin K (Bethyl cat# A301-939A, 1:500); cyclin H (Bethyl cat# A301-674A, 1:1000); PARP (Cell Signaling cat# 9542, 1:1000); and α-Tubulin DM1α (Sigma cat# T9026, 1:5000). The following ChIP-grade antibodies were used for ChIP-seq (10 µg each): Pol II (Santa Cruz cat# sc-899); CDK12 (gift of Arno Greenleaf); Pol II CTD Ser-2 (Millipore cat# 04-1571); and H3K27Ac (Abcam cat# AB4729).

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).

HAP1 or Jurkat cells were lysed in RIPA buffer (50mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS, pH 7.4 ± 0.2) with Protease Inhibitor (Roche), Phosstop Phosphatase Inhibitor (Roche), and 2.5 U/ml Universal Nuclease for Cell Lysis (Pierce) by incubating on ice 30 min. The lysates were clarified by spinning at 21,000 g for 30 min at 4 °C and the concentration of the lysate was determined using BCA protocol (Pierce). Primary antibodies in this study include: CDK7 (Cell Signaling Technology, 2916, 1:1,000), Cyclin H (Bethyl Labs, A301-674A , 1:1000), Cyclin K (Bethyl Labs, A301-939A, 1:1000), CDK2 (Bethyl Labs, A301-812, 1:1,000), Phospho-CDK2 T160(Cell Signaling Technology, 2561, 1:1000) CDK1 (Bethyl Labs, A303-663, 1:1,000), Phospho-CDK1 T161 (Cell Signaling Technology, 9114, 1:1000)Tubulin (Cell Signaling Technology, 3873 1:5,000), PARP (Cell Signaling Technology, 9542, 1:2,000), Phospho-CTD Ser2 (Millipore, 04-1571, 1:2,000), Phospho-CTD Ser5 (Millipore, 04-1572, 1:5,000), Phospho-CTD Ser7 (Millipore, 04-1570-I, 1:1000), Total Pol II (Santa Cruz Biotechnology, sc-899, 1:200). Secondary antibodies were infra-red labeled antibodies (LI-COR, used at 1:10,000) and blots were imaged on an Odyssey CL_Ximager.