Infra red labeled antibodies

MOLT4 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 minutes. The lysates were clarified by spinning at 21,000g for 30 minutes at 4°C and the concentration of the lysate was determined using BCA protocol (Pierce). Primary antibodies in this study include: CDK9 (Cell Signaling Technology, 2316), CDK7 (Cell Signaling Technology, 2916), CDK2 (Bethyl Labs, A301-812), CDK1 (Bethyl Labs, A303-663), Cereblon (Novus Biologicals, NBP1-91810), Tubulin (Cell Signaling Technology, 3873), PARP (Cell Signaling Technology, 9542), MCL-1 (Santa Cruz Biotechnology, sc-819), Caspase-3 Full Length (Cell Signaling Technology,9668), Caspase-3 Cleaved (Cell Signaling Technology, 9661), ɣH2A.X (Cell Signaling Technology, 9718), Phospho-CTD Ser2 (Millipore, 04-1571), Phospho-CTD Ser5 (Millipore, 04-1572), Total Pol II (Santa Cruz Biotechnology, sc-899), Cyclin T1 (Cell Signaling Technology, 8744), CDK10 (Cell Signaling Technology, 36106), ENL (Cell Signaling Technology, 14893), AFF4 (Abcam, ab103586), and ELL (Cell Signaling Technology, 14468S). Secondary antibodies used were infra-red labeled antibodies (LI-COR) and blots were imaged on an Odyssey CL_X imager.

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.