Loading control gapdh

Cell pellets corresponded to control (cells + DMSO), BaP-treated (1 µM), and co-treated (DAS 60 µM and 600 µM combined with BaP 1 µM) cells after 24-h treatment. A protease inhibitor cocktail was mixed with the lysis buffer, and the combination was added to each pellet. The total protein expression was determined using Western analysis. Samples containing 0.6 mg/mL of protein and 1:125 dilution of primary antibody were used. The microplate was loaded according to ProteinSimple’s protocol and placed in the instrument, along with the capillary responsible for the reaction. DNA Polymerase β (Abcam #ab26343) and loading control GAPDH (D16H11; Cell Signaling #5174S) were the specific primary antibodies tested. The protein was identified, the chemiluminescence reaction was determined, and the digital blot image was taken.

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).