Rabbit anti cdk9

293_HFL cells were plated at 5×10⁶ cells per 75cm² flask (#229341, cell treat scientific) in 15ml of complete DMEM media. After 24 hours, cells were treated with indicated compounds for 2h. After the treatments, cells were harvested and washed three times with 1xPBS. Halotag-FKBP Immunoprecipitation was performed using Halo-Trap Agarose beads (# OTA-200 Chromotek) as per manufacturer instructions with some modifications. Briefly, HFL cell pellet was resuspended in 500μL of 1X lysis buffer (#9803, Cell Signaling) for 30min at 4°C, vortex every 10 min, and then the suspension was pelleted at 14000 × g for 10 minutes at 4°C. The supernatant was precleared with 25 μL of lysis-buffer-washed binding control agarose beads (#bab-20, chromotek) rotating at 4°C for 1hr. Non-specific aggregates were removed by centrifugation at 2500 × g for 5 mins and 5% of the supernatant was used as the input. Remaining supernatant was incubated with 25 μL of lysis-buffer-washed Halo-Trap agarose beads rotating at 4°C for 1 hr. Halo-trap beads were washed thrice with 500 μL of lysis buffer and fractionated by SDS-PAGE. Immunoblotting was done using mouse-anti FKBP (#sc-136962, Santa cruz), rabbit-anti BRD4 (#13440S, Cell Signaling), mouse-anti Plk1 (#17056, Abcam) and rabbit-anti CDK9 (#2316, Cell signaling), rabbit-anti phospho-Rpb1 CTD Ser2 (#13499, Cell Signaling)