Rabbit anti gapdh

ZR75 cells were seeded in 100 mm petri dishes at a density of 2,000,000 cells/dish. Cells were treated with TZ, BMS-202, or a combination of both for 48 hours. Cell lysates were collected, and 30 μg of proteins were resolved on 10% polyacrylamide SDS PAGE gels and then transferred onto PVDF membranes. Membranes were probed with the following primary antibodies: anti-rabbit Akt (CST: 9272S), anti-rabbit phospho-Akt (Ser473) (CST: 4060S), anti-rabbit mTOR (CST: 2983S), anti-rabbit phospho mTOR (S2448) (Abcam: ab109268), anti-mouse ErbB2 (Abcam: ab16901), anti-rabbit phospho ErbB2 (Abcam: ab53290), and anti-rabbit vimentin (CST: 46173S). Anti-rabbit GAPDH (Cell Signaling: 8480S) was used to ensure equal loading of protein samples. Blots were incubated with ECL Western blotting substrate (Pierce Biotechnology, Rockford, IL, USA) and chemiluminescence was recorded using the iBrightTM CL1000 imaging system (Thermo Fisher Scientific, Wal-tham, MA, USA). Quantification was done using ImageJ software.

Cells were cultured and treated according to Fig 1. Cells were harvested and lysed on ice for 15min in 1xRIPA buffer (Alfa Aesar, catalog number J60629) containing 1x protease inhibitor cocktail (Thermo Scientific, catalog number 87786). 100ug of cell lysate boiled at 95°C for 15 min in Laemilli buffer (10% glycerol, 2% SDS, 0.1% bromophenol blue, 200mM Tris HCl pH 6.8, 20mM DTT). Samples were run on an SDS-PAGE gel and transferred to a polyvinyl difluoride (PVDF) membrane. Prior to transfer, the membrane was soaked in methanol for 2 minutes, then washed with Transfer Buffer (0.3%w/v Tris, 1.45% w/v glycine, and 10% v/v methanol). Anti-rabbit ICAM-1(Cell Signaling, catalog number 49155) and anti-rabbit GAPDH (Cell Signaling, catalog number 21185) were used to probe the membrane at 1:500 and 1:5000, respectively. An anti-goat, anti-rabbit horseradish peroxidase-linked secondary antibody (1:10,000, Jackson Labs, catalog number 111-035-144) followed by detection using SuperSignal™ Chemiliuminescent HRP Substrate and exposure to film. ImageJ software was used to compare protein levels normalized to GAPDH.

Total proteins were extracted from HUVECs with radioimmune precipitation assay buffer (Solarbio), and the amount of protein was measured with a BCA kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Equal amounts of protein were separated via 12% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, and then transferred onto polyvinylidene fluoride membranes (Millipore). The membranes were then blocked with 5% milk for 1 h and incubated overnight at 4°C with primary antibodies, including anti‐rabbit‐Bcl‐2 (ab59348; Abcam), anti‐rabbit Bax (2772S; Cell Signaling Technology), and anti‐rabbit GAPDH (5174; Cell Signaling Technology). The next day, the membranes were washed three times with TBST buffer and then incubated with horseradish peroxidase‐labeled secondary antibodies for 2 h. Finally, protein bands were visualized with electrochemiluminescence (ECL) luminescent solution.