Whole-cell extracts were prepared in lysis buffer [50 mM tris (pH 7.5), 150 mM sodium chloride, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 1× Roche protease inhibitor cocktail, and 1× Roche PhosSTOP phosphatase inhibitor cocktail], and protein concentration of each sample was quantified using a Pierce BCA protein assay kit (Thermo Fisher Scientific). Extracts were diluted in SDS loading buffer [50 mM tris (pH 6.8), 2% SDS, 10% glycerol, 0.1% bromophenol blue, and 50 mM dithiothreitol (DTT)], and total protein (20 to 30 μg) was separated according to mass using SDS–polyacrylamide gel electrophoresis. Proteins were transferred to polyvinylidene difluoride membrane and incubated with the appropriate antibody at the manufacturers’ recommended dilution. Primary antibodies used were eIF2α (CST, no. 9772), p-eIF2α (Ser51) (Abcam, no. 32157), p–4E-BP1 (Ser65) (CST, no. 9456), 4E-BP1 (CST, no. 9644), p–p70 S6K (Thr389) (CST, no. 9205), p70 S6K (CST, no. 2708), p-Akt (Ser473) (CST, no. 4058), Akt (CST, no. 9272), p53 (Dako, no. M7001), Sestrin2 (Abcam, ab178518), p-ATM (Ser1981) (Abcam, no. 81292), ATM (Abcam, no. 32420), p-Chk2 (Thr68) (CST, no. 2661), Chk2 (no. 2662), GCN2 (CST, no. 3302), PKR (CST, no. 12297), PERK (CST, no. 3192), ATF4 (CST, no. 11815), PP6c (Abcam, no. 131335), TSC2 (CST, no. 4308S), BiP (Proteintech, no. 11587-1-AP), C/EBP homologous protein (CHOP) (Proteintech, no. 15204-1-AP), and β-tubulin (CST, no. 2146).

For enhanced chemiluminesence (ECL) detection, membranes were incubated with horseradish peroxidase–conjugated α-mouse (Dako, no. P0447) or α-rabbit (GE Healthcare, no. NA934V) secondary antibodies. Signal was developed by incubating membranes in ECL Prime Solution for 5 min, and x-ray film was exposed to the membrane to visualize luminescence. For fluorescent detection, membranes were incubated with IRDye Light-labeled α-mouse (CST, no. 5257S) or α-rabbit (CST, no. 5366S) secondary antibodies. Fluorescent signal was detected using LI-COR Odyssey Imaging Systems (LI-COR Biosciences), and images were analyzed with LI-COR Image Studio software (package version 5.2.5). Quantification of signal from both ECL- and LI-COR–detected blots was carried out using LI-COR Image Studio. Signal for a phosphorylated protein was normalized to that for the total abundance of the corresponding protein.

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