P akt ser473 9271

Rictor (sc-271081) and (β-) tubulin (sc-55529) antibodies were purchased from Santa Cruz (Santa Cruz, CA). p-Akt Ser473 (#9271) and Akt1 (#2967) antibodies were obtained from Cell Signaling Tech (Danvers, MA). Puromycin was purchased from Sigma (Shanghai, China). All the cell culture reagents were obtained from Gibco (Shanghai, China).

Frozen liver fragments (50 mg) were homogenized in 500 μL of homogenization buffer: [7M Urea, 2M Thiourea, 4% CHAPs, 40 mM DTT, supplemented with protease and phosphatase inhibitors (Complete and Phostop, respectively; Roche, Mannheim, Germany)] and ultracentrifuged at 75.000 rpm for 45 min. Supernatants were collected in a clean cryo-tube and conserved in -80°C until use. Equivalent amounts of protein were separated in denaturing polyacrylamide gels and transferred onto a nitrocellulose membrane. The primary antibodies against p-eIF2α (Ser51-#9721), IRE1α (#3294), Irβ (#3025), AKT(#9272), p-AKT (Thr308-#9275) and p-AKT (Ser473-#9271) were purchased from Cell Signaling Technology, XBP1(sc7160) and EIF2α (sc11386) from Santa Cruz Biotechnology and ATF6 (IMG273) from IMGENEX, alpha-tubulin (T6074) from Sigma. Detection of immunolabeled proteins was performed using a commercial chemiluminescent assay (ECL prime; Amersham, Buckinghamshire, UK). Visualization and quantitative measurements were made with a CCD camera and software for Western blot image analysis (Odissey Fc Imager System and Image Studio Lite v 4.0, respectively; Li-COR, Bad Homburg, Germany).