Anti pcdk9

C11 cells were adjusted to 1 × 10⁶ cells/ml and stimulated with 1 μM prostratin or 0.05 μM EK-16A for various time. Whole-cell extracts or the nuclear fractions were prepared as described previously^{99 (link), 100 (link)}. Proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane, and visualized with primary and secondary antibodies and ECL chemiluminescence system (Santa Cruz Biotechnology, Santa Cruz, CA). The following primary antibodies were used: anti-IκBα (catalog # 9242), anti-p-IκBα (# 9246), anti-CDK9 (# 2316), anti-p-CDK9 (# 2549), anti-Cyclin T1 (# 8744), anti-α-Tubulin (# 2125), anti-TBP (# 12578) (Cell Signaling, Danvers, MA), and anti-p65 (catalog # sc-56735; Santa Cruz).

Anti–phospho-Histone γ-H2AX (Ser139) was from Millipore (Billerica, MA); MDC1 and 53BP1 antibodies were gifts from Dr. Zhenkun Lou, Mayo Clinic. Anti-Bora was obtained from New England Peptide (Gardner, MA). Anti–HA, GST, anti-PLK1 as well as anti-pCDK9 and CDK9 were from Cell Signaling Technology, Inc (Danvers, MA); anti–FLAG and actin antibodies were purchased from Sigma-Aldrich, Inc. (St. Louis, MO); and the horseradish peroxidase–conjugated secondary antibodies against mouse and rabbit were obtained from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). Fluorescent dye–conjugated secondary antibodies were obtained from Invitrogen Corp (Carlsbad, CA).

Treatment of 5 × 10⁵ A673, SKNMC or TC-71 cells with 500 nM JQ1, 100 nM CDKI-73, or the combination of both; DMSO was used as solvent control. Cells were washed once with PBS and harvested in lysis buffer containing 50 mM Tris HCl, 10 mM beta-glycerophosphate, 150 mM NaCl, 1% Triton-X 100, 5 mM Na₄PO₇, 1 mM Na₃VO₄, 50 mM NaF supplemented with protease inhibitor (Complete mini, Roche Diagnostics). Protein concentration was determined by BCA (Thermo Fisher Scientific, Waltham, MA USA). Ten to thirty micrograms of protein extract were resolved on 4%–12% SDS-PAGE and transferred onto a nitrocellulose membrane (Thermo Fisher Scientific).
Primary antibodies were used as follows: anti-BRD4 rabbit monoclonal antibody (ABCAm, Cambridge, USA), anti-FLI1 monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA), anti-pCDK9 (Cell Signaling Technology), anti-PARP rabbit polyclonal antibody (Cell Signaling Technology), anti-Caspase7 antibody (Cell Signaling) and loading control GAPDH (Cell Signaling Technology). After incubation with the appropriate secondary peroxidase-conjugated antibodies, detection was performed with the ECL chemiluminescence reagent (Amersham Biosciences, Little Chalfont, UK) or SuperSignal West Femto (Thermo Fisher Scientific).