Western Blot Analysis of Protein Lysates

Cells were lysed in radioimmunoprecipitation assay lysis (RIPA) buffer. The cell lysate was collected after centrifugation at 13,000× g for 15 min. Proteins (20 μg) from cell lysates were separated by 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (BiotraceTM, PALL Life sciences, Ann Arbor, MI, USA (BSP0161)). Signals after immunoblotting with primary and secondary antibodies overnight on the sample membrane were detected by a chemiluminescence kit (Immobilon Western Chemiluminescence HRP Substrates, Merck-Millipore, Billerica, MA, USA (WP20005)). A luminescence/fluorescence imaging system (GE Healthcare) and multi-gauge image analysis software version 3.0 (Fujifilm, Stockholm, Sweden) were used to analyze the image. The preparation of the RIPA buffer and detailed procedure has been described in a previous study [27 (link)]. The antibodies against α-tubulin (62204), p-eIF2α (44728G), eIF2α (AHO0802) (InvitrogenTM, Thermo Fisher Scientific, Carlsbad, CA, USA), ATF4 (Proteintech, Rosemont, IL, USA (10835-1-AP)), and xCT (Cell Signaling Technology, Beverly, MA, USA (#12691)) were used in this study.