Ab78318

Cells were seeded at 1,000,000 cells/well/2 mL into 6-well plates. After an overnight incubation, cells were treated with GS-626510 for indicated time points for Western blot analysis. Cells were washed with ice-cold PBS post treatment and resuspended in 200 μL with RIPA buffer containing Protease and Phosphatase Inhibitor (ThermoFisher Scientific, cat#78430). Proteins (10 μg) were resolved using SDS-polyacrylamide gels and blotted onto nitrocellulose membranes. Membranes were washed with TBS (140 mmol/L NaCl, 50 mmol/L Tris-HCl; pH 7.2) containing 0.1%Tween20 (TBST) and 5% skimmed milk to block nonspecific protein binding. Membranes were incubated with antibody against c-Myc (1:1000, 5605S, Cell Signaling Technology), BRD4 (1:500, ab128874, Abcam), c-Myc-phsphoT58 (1:500, ab28842, Abcam), c-Myc-phospho S62 (clone 33A12E10) (1:500, ab78318, Abcam), or GAPDH (1:10000) in TBST for overnight at 4°C, washed three times with TBST, and then incubated with the fluorescently-labeled secondary antibody (1:10000) for 1 hour at room temperature. Immunoreactive proteins were scanned and quantified using Odyssey fluorescence imaging system.

Pancreatic cancer cells were plated in DMEM with 2% FBS and 24 hours later treated with either Vehicle, 10μM DT1154, 0.5 μM INK128, or the combination of DT1154 and INK128 for 6 hours. Cells were lysed, run on an SDS page gel, and quantified using a LI-COR Odyssey as previously described (23 (link)). For tumor cell lysates, 50–150mg of tumor tissue was homogenized, lysed, and run as above. For the phosphokinase array (R&D Systems), PANC89 cells were treated and lysed as above and then 450 μg of protein per membrane was used per the kit protocol. Details of the MYC immunoprecipitation can be found in the Supplemental Methods. The following antibodies were used for western blot analysis: c-MYC (Y69 ab32072, Abcam; N-262 SC-764, Santa Cruz Biotechnology), pS62 MYC (ab78318, Abcam), pT58 MYC (Y011034, Applied Biological Materials),‎ pAKT (CS4060, Cell Signaling Technology), AKT (CS2920, Cell Signaling Technology), pPRAS40 (CS2997, Cell Signaling Technology), PRAS40 (CS2691, Cell Signaling Technology), pS6 (CS4858, Cell Signaling Technology), 4EBP1 (CS9644, Cell Signaling Technology), GAPDH (AM4300, Applied Biosystems), IRDye800 (Rockland), and Alexa Fluor 680 (Molecular Probes). GAPDH and secondary antibodies were used at 1:10,000 and all other primary antibodies were used at 1:1000.