Ab184551

Proteins were extracted from cells using ice-cold RIPA buffer (Beyotime, Shanghai, China) supplemented with protease inhibitors and phosphase inhibitors. The protein concentrations were measured by BCA method. Then 20 μg of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a 0.45 μm PVDF membrane. The membrane was blocked with 5% skim milk for 1 h and incubated with primary antibodies overnight. After washing with PBS, blots were incubated with secondary antibodies. Images were acquired by enhanced chemiluminescence. GAPDH antibody was used as a control. Results were treated with gray analysis by Gel-Pro Analyzer (United States Biochemical, Cleveland, OH). The semi-quantitative analysis was performed according to the relative expression of objective protein and GAPDH.
The primary antibodies used in this study were as follow: anti-CAV1 (1:1000, Rabbit polyclonal antibody, ab18199, Abcam, Cambridge, MA, USA); anti-AKT (1:500, Mouse monoclonal antibody, #9272, Cell Signaling Technology, Beverly, MA, USA); anti-p-AKT (1:1000, Mouse monoclonal antibody, Ser473, 66,444–1-Ig, Proteintech, Manchester, UK); anti-Cyclin D1 (1:1000, Rabbit monoclonal antibody, ab16663, Abcam); anti-P70 (1:1000, Rabbit monoclonal antibody, ab184551, Abcam); anti-GAPDH (1:500, Mouse monoclonal antibody, ab8245, Abcam).

After 48 h of transfection, the cells were lysed with RIPA lysate (Pierce, USA) in ice for 10 min, and the total protein was extracted. The concentration of protein was measured with the BCA protein assay kit (Pierce, USA). The protein samples were separated by SDS polyacrylamide gel electrophoresis and transferred into polyvinylidene difluoride membranes (PVDF; Millipore, Billerica, MA). The membranes were blocked with 5% skim milk for 1 h at room temperature and incubated overnight at 4°C with primary antibodies. The primary antibodies are as follows: anti-Bax (50599-2-Ig), anti-Bcl2 (12789-1-AP), anti-caspase3 (19677-1-AP), anti-p53 (60283-2-Ig), anti-GAPDH (60004-1-Ig), anti-AKT (10176-2-AP), anti-p-AKT (Ser473, 66444-1-Ig), anti-mTOR (66888-1-Ig), and anti-p-mTOR (Ser2448, 66888-1-Ig) were obtained from PTG, and anti-p70 (ab184551), anti-p-p70 (Ser371, ab109393), and anti-TRIB1 (ab137717) were obtained from Abcam. Following this, the secondary antibody (HRP-conjugated Goat Anti-Rabbit IgG, SA00001-2; and HRP-conjugated Goat Anti-Mouse IgG, SA00001-1, PTG) was applied and incubated with the membrane for 1 h at room temperature. Finally, the proteins were visualized with enhanced chemiluminescence reagents (Pierce, USA). The QUANTITY ONE software (Bio-Rad Laboratories, Inc.) was used to scan and calculate the relative expression quantity of each protein.

The extracted total protein was subjected to SDS-PAGE and transferred onto PVDF membranes (Millipore, Bedford, MA, USA). Following blockade, membranes were treated with primary antibodies overnight and with secondary antibody for 2 h in the dark. Primary antibodies against GLI1 (ab49314), SOX2 (ab93689), MYC (ab9106), VEGF (ab32152), β-catenin (ab32572), MMP2 (ab215986), p70S6K2 (ab184551), GSK3β (ab93926), p-GSK3β (Y216) (ab75745), p-GSK3β (S9) (ab75814), Cyr61 (ab24448), and GAPDH (ab8245), were all from Abcam (Cambridge, MA, USA). All samples were assayed in triplicate.