P pi3k p85 tyr458

Total protein lysates were extracted from cells and equal amounts of total protein were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Nonspecific binding was blocked with 5% milk in tris-buffered saline with Tween 20, and membranes were incubated overnight at 4°C with primary antibodies against the following proteins: CIP2A (1 : 1000), AKT1 (1 : 2000), phosphorylated AKT1 (p-AKT1) (Ser473) (1 : 1500), PI3K p85 (1 : 500), p-PI3K p85 (Tyr458) (1 : 1000), mTOR (1 : 1000), p-mTOR (Ser2448) (1 : 2000), and poly(ADP-ribose) polymerase (PARP, 1 : 1500) (all from Cell Signaling Technology, Danvers, MA, USA). Membranes were then incubated with horseradish peroxidase-conjugated secondary antibody and protein bands were detected by enhanced chemiluminescence (Thermo Scientific, Waltham, MA, USA).

Rabbit anti-CLDN1 (51-9000) and anti-CLDN2 (51-6100) antibodies were purchased from Thermo Fisher Scientific (Rockford, IL, USA). Rabbit anti-Akt, anti-p-Akt (Ser473), PI3K p85, p-PI3K p85 (Tyr458), and anti-ERK1/2 antibodies were from Cell Signaling Technology (Beverly, MA, USA). Mouse anti-p-Stat3 (pY705), anti-Stat3, and anti-p38 antibodies were from BD Biosciences (San Jose, CA, USA). Rabbit anti-p-Akt (Thr308), anti-p-ERK1/2, and goat anti-β-actin antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). LY and MDC were from Tokyo Chemical Industry (Chuo-ku, Tokyo, Japan). C3G, CHL, and MβCD were from FUJIFILM Wako Pure Chemical Industries (Chuo-ku, Osaka, Japan). CAN, Cyanidin, mouse anti-ZO-1 antibody, P3G, rabbit anti-p-p38 antibody, and SB203580 were from Cayman Chemical Company (Ann Arbor, MI, USA), Tokiwa Phytochemical (Sakura-shi, Chiba, Japan), Zymed Laboratories (South San Francisco, CA, USA), Toronto Research Chemicals (North York, ON, Canada), Cusabio (Houston, TX, USA), and AdipoGen (San Diego, CA, USA), respectively. All other reagents were of the highest grade of purity available.