Western Blot Analysis of Protein Signaling

After incubation and pharmacological treatments as indicated, cells were rapidly cooled by washing with ice-cold PBS and then lysed in 2X-Laemmlli Sample buffer (0.5 M Tris pH 6.8, glycerol, 10% SDS) supplemented with 1 mM sodium orthovanadate, 10 mM okadaic acid, and 20 mM of protease inhibitor. Whole-cell lysates were then syringed 5 times with a 27.5-gauge syringe. Lysates were then heated at 65 °C for 15 min under reducing conditions (supplementation of lysis buffer with 10% beta-mercaptoethanol and 5% bromophenol blue), resolved by SDS-PAGE, and transferred to 0.2 m pore PVDF membrane (PALL Life Science). The membrane was blocked with 3% bovine serum albumin (BSA) and then incubated with indicated antibodies in 1% BSA at 1:1000 dilution at 4 °C overnight. The membrane was then subjected to a secondary horseradish peroxidase–conjugated antibody (with 1% BSA) and left to shake for 1 h at RT before imaging. Bands were visualized with Luminata ECL chemiluminescence substrate (Milipore Sigma).