Anti p 4e bp1 t37 46

Cells were lysed in lysis buffer including 0.2% protease inhibitor cocktail III (Calbiochem, San Diego, CA; La Jolla, CA, USA). After homogenization, the cell lysates were incubated on ice for 30 min and centrifuged at 12,000 g for 10 min. The amount of total protein was measured by protein assay kit (Bio-Rad, Hercules, CA, USA), and the proteins were then mixed with SDS sample buffer and boiled for 5 min before loading into a 10% or 8% SDS-PAGE gel (Bio-Rad, Hercules, CA, USA). The proteins were transferred onto nitrocellulose membrane after electrophoresis. The blots were blocked and then incubated with primary antibody followed by a horseradish peroxidase (HRP)-conjugated secondary antibody. Immunoreactive bands were visualized by enzyme-linked chemiluminescence with an ECL kit. The primary antibodies were as follows: anti-MELK (ab108529), anti-α-tubulin (ab7291), and anti-mTOR (ab2732) from Abcam (Cambridge, UK); anti-4E-BP1 (#9644), anti-p-4E-BP1 (T37/46) (#2855), anti-S6 (#2317), anti-p-S6 (S235/236) (#4858), anti-p-PRAS40 (Thr246) (#13175), anti-p-PRAS40 (Ser183) (#5936), and anti-raptor (#2280) from Cell Signaling Technology (Danvers, MA, USA); and anti-Flag from Sigma (#3165, St Louis, MO, USA). The immune complex was detected using HRP-conjugated secondary antibodies (ZSGB-BIO, Beijing, China). Rapamycin was purchased from Sigma (Solon, OH, USA).