Pcdk9

Mia^R cells were grown in 2D, 10 cm dishes and treated with 300 nM UNC-721A dissolved in DMSO for times ranging from 1 to 4 h. The samples were harvested using MIB-MS lysis buffer (above), and determination of protein levels was performed by Bradford analysis as described above. The proteins amounts were normalized and the samples were mixed with 4× sample buffer and 20 μg of each sample applied to sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis. The proteins were transferred to polyvinylidene difluoride (PVDF) and the membrane blocked with 5% bovine albumin in Tris-buffered saline with 0.05% Tween-20 (TBST) for 1 h at room temperature. Primary antibodies for MCL-1 (Santa Cruz, Santa Cruz, CA), pSRSF (EMD Millipore, Darmstadt, Germany), tSRSF1 (Thermo Fisher, Waltham, MA), tSRSF6 (Invitrogen, Waltham, MA), PARP (Cell Signaling, Danvers, MA), CDK9 (Santa Cruz), pCDK9 (Cell Signaling), RNA polymerase II (phospho Ser2, Abcam, Cambridge, UK), and β-Actin (Santa Cruz) were diluted in 5% bovine serum albumin (BSA)/TBST, and the membranes were incubated overnight at 4 °C. The membranes were washed three times with TBST, followed by incubation in horseradish peroxidase (HRP)-conjugated secondary antibodies (Promega) in 5% nonfat dry milk in TBST. The blots were imaged using the ChemiDoc Touch Imaging System and ECL reagent (Bio-Rad).