Glyceraldehyde 3 phosphate dehydrogenase gapdh sc 47724

The PARPi olaparib (AZD2281) was purchased from Selleckchem (Houston, TX, USA). Doxorubicin was purchased from Sigma-Aldrich (St. Louis, MO, USA). All reagents, including carboplatin (Dong-A ST, Seoul, Korea), cisplatin (JW Pharmaceutical, Seoul, Korea), and paclitaxel (Samyang Biopharm, Gyeonggi-do, Korea) were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich). MTT was purchased from Amresco (Solon, OH, USA). Primary antibodies used in this study included the following; anti-cleaved caspase 3 (D175, #9661; Cell Signaling Technology; Danvers, MA, USA), phospho-histone H2AX (Ser 139, sc-517348), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, sc-47724; Santa Cruz Biotechnology; Dallas, TX, USA), and anti-β-actin (A5441; Sigma-Aldrich) antibodies.

The total cell lysates were prepared in a high KCl lysis buffer with a complete protease inhibitor cocktail (Roche, Switzerland). The protein concentration was determined using a BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Equal amounts of protein samples were subjected to SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA). The membranes were treated with 1% blocking solution in TBS for 1 h, and immunoblots were probed with the indicated primary antibodies at 4°C overnight. The primary antibodies used were P4HA2 (ab233197), Collagen I (ab34710), Collagen III (ab7778), and Collagen IV (ab6586) from Abcam (Cambridge, UK); Twist1 (25465-1-AP) from Proteintech Group (Rosemont, PA, USA); Snail (#3879), Slug (#9585), AKT (#4691), phosphor-AKT (#4060), PI3K(#4257), and phospho-PI3K(#4228) from Cell Signaling Technology (Danvers, MA, USA); and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (sc-47724) from Santa Cruz Biotechnology (Dallas, TX, USA). Then, the membranes were washed and incubated with HRP-labeled secondary antibodies (Molecular Probes, MA, USA). The fluorescence signals were detected using a BM Chemiluminescence Western Blotting kit (Roche, Basel, Switzerland). Densitometry quantification was calculated from triplicate assays and analyzed using ImageJ software.