Ab32111

Protein (50 mg per sample) was loaded per line and separated on 8%–15% tris-glycine polyacrylamide gels and transferred to nitrocellulose membranes. Immunoblots were blocked for 2 h in Tris-buffered saline Tween-20 (TBST, 20 mM Tris-HCl, 150 mM NaCl, pH 7.5, and 0.05% Tween 20) containing 5% skim milk. The blots were then incubated with primary antibodies in TBST at 4°C overnight. Membranes were washed three times with TBST and then incubated with secondary antibody for 2 h at room temperature. Membranes were washed again and developed with enhanced chemiluminescence (ECL) and detected with X-ray films. The blots were visualized with ImageQuant TL 1D (GE Healthcare, USA). Primary antibodies used from Cell Signaling Technologies (Danvers, MA, USA) were as follows: p-Tyr452 Gab2 (#3882, 1 : 1000), PI3 kinase p85 (#4257, 1 : 1000), Akt (#9272, 1 : 1000), p-Ser473Akt (#4058, 1 : 1000), p-Ser9GSK3β (#9323, 1 : 1000), and beta-actin (#3700T, 1 : 2000). Primary antibody directed against Grb2 (Abcam PLC, Cambridge, UK, #ab32111, 1 : 1000), GSK3β (Abcam PLC, Cambridge, UK, #ab131356, 1 : 1000), and Gab2 (Santa Cruz, USA, sc-365590, 1 : 500) were also used in the experiments. Secondary antibodies were goat anti-rabbit (BA1054, 1 : 2000) (Boster Biological Technology Co. Itd, Wuhan, China) or anti-mouse (BA1050, 1 : 2000).

Primary antibodies p-Tyr452 Gab2 (#3882, 1 : 1000), PI3 kinase p85 (#4257, 1 : 1000), Akt (#9272, 1 : 1000), p-Ser473Akt (#4058, 1 : 1000), p-Ser9GSK3β (#9323, 1 : 1000), and beta-actin (#3700T, 1 : 2000) were purchased from Cell Signaling Technologies (Danvers, MA, USA). Primary antibody directed against Grb2 (#ab32111, 1 : 1000) and GSK3β (#ab131356, 1 : 1000) were from Abcam PLC (Cambridge, UK). Primary antibody Gab2 (sc-365590, 1 : 500) was from Santa Cruz (Santa Cruz, USA). Secondary antibodies were goat anti-rabbit (BA1054, 1 : 2000) (Boster Biological Technology Co. Itd, Wuhan, China) or anti-mouse (BA1050, 1 : 2000).

The transfected cells were collected after 24 h, and the cells were lysed using RIPA lysis buffer in order to extract total protein. The protein concentration was determined using the BCA method. Proteins were then separated by 10% SDS-PAGE gel electrophoresis, and transferred to a membrane, blocked with 5% skimmed milk powder at room temperature for 1 h. The membranes were then incubated with primary antibodies targeting Pax4 (PA1-108, ThermoFisher), Grb2 (ab32111; Abcam), p-Raf (ab157201; Abcam), T-Raf (ab230850; Abcam), p-MEK (ab278562; Abcam), t-MEK (ab32576; Abcam), p-ERK (ab136926; Abcam), t-ERK (ab184699; Abcam), Vimentin (ab92547; Abcam), Bax (ab32503; Abcam), CyclinD1 (ab16663; Abcam) and GAPDH (ab181602; Abcam). The membranes were incubated at 4°C overnight, washed with TBST three times, five min each time. Then, the membranes were incubated with secondary antibodies for 1 h at room temperature. The membranes were washed with TBST three times, five min each time. Finally, ECL luminescent solution was added to the dark room, exposed and developed.