Ecl prime chemiluminescence detection system

The cells were lysed in RIPA buffer (Sigma) that contained 150 mM NaCl, 1.0% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0. Protein lysates were then separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore). The membranes were then blocked with 10% non-fat milk in Tris-buffered saline with 0.1% Tween 20 (TBST). After blocking, the membranes were incubated overnight at 4°C with primary antibodies: anti-β-DG (1:500; ab49515, Abcam), horseradish peroxidase (HRP)-GFP (1:1000; LS-C50850, LifeSpan BioSciences), mouse anti-α-DG (1:1500; 05-593; Millipore), and anti-β-actin (1:10000; A5441, Sigma) diluted in 5% non-fat milk in TBST or anti-GFP (1:1000; MAB3580, Chemicon) and anti-Cdc42 (1:500; 11A11, Cell Signaling) diluted in 5% bovine serum albumin in TBST. The blots were washed three times with TBST followed by 1 h incubation with peroxidase-conjugated secondary antibody, diluted 1:5000 in TBST that contained 5% non-fat milk. After washing, bands were detected using the ECL PRIME chemiluminescence detection system (GE Healthcare).