Rabbit monoclonal anti human gapdh

Cells and tumor tissues were lysed with protein extraction reagent (KeyGEN BioTECH, Jiangsu, China) according to the manufacturer’s instructions, and total cell lysate protein samples were obtained. Then samples were equally loaded on 10% SDS-polyacrylamide gel, electrophoresed, and transferred to polyvinylidene difluoride membranes (Bio-Rad). After blocking with 5% BSA in Tris-buffered saline (TBS) with 0.1% Tween 20 (TBST) for 2 hr, the membranes were incubated with the primary antibodies rabbit-polyclonal anti-human DUSP8 (1:2,000; Abcam, Cambridge, UK), rabbit-monoclonal anti-human ERK (1:1,000; Cell Signaling Technology (Danvers, MA, USA)), rabbit-polyclonal anti-human p-ERK (1:2,000; Cell Signaling Technology), rabbit-monoclonal anti-human AKT (1:1,000; Cell Signaling Technology), rabbit-monoclonal anti-human p-AKT (1:2,000; Cell Signaling Technology), or rabbit-monoclonal anti-human GAPDH (1:2,000; Cell Signaling Technology) at 4°C overnight. After the overnight incubation with the primary antibodies, membranes were washed in TBST three times and subsequently probed with a secondary anti-rabbit Ab-conjugated to horseradish peroxidase (HRP) for 1 hr (1:2,000; Cell Signaling Technology). Finally, the signals were detected and analyzed using the chemiluminescence image system (Bio-Rad).