Multigauge image analysis software version 3

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.

The cellular protein was prepared by the collection of supernatant of RIPA lysis buffer after scraping and centrifugation at 13,000× g at 4 °C for 15 min. The protein concentration was analyzed by Bradford reagent (Bio-Rad, Hercules, CA, USA). The sample proteins (20–30 μg) were heated at 110 °C for 10 min and separated by electrophoresis (8–15% SDS-polyacrylamide gel electrophoresis). After protein separation, the proteins were transferred onto the polyvinylidene difluoride membranes (PVDF, Biotrace™, PALL Life sciences, Ann Arbor, MI, USA). Sample-PVDF sample membrane was blocked with 5% skimmed milk at room temperature for 1 h. The specific primary/secondary antibodies, enhanced chemiluminescence reaction, and luminescence fluorescence image capture system (GE healthcare) were used to observe specific proteins. The sample band was analyzed by MultiGauge image analysis software version 3.0 (Fujifilm, Stockholm, Sweden). The TID-1 L/S antibody (sc-18819) was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). The galectin-7 antibody (Cat. No. PA5-31866) was obtained from Ambion™, Thermo Fisher Scientific (Eugene, OR, USA). The α-tubulin antibody was obtained from Invitrogen™, Thermo Fisher Scientific (Eugene, OR, USA). Chase analysis was performed by adding the cycloheximide (100 μg/mL) for the indicated period and analyzed by Western blot.

A quantity of 7.0 × 10⁵ cells was seeded in a 6 cm dish and the cultured medium with serum-free medium was collected for 40 h. The supernatant of the collected culture medium was centrifuged at 4 °C 12,000× g for 10 min. The 5–10 μg samples were mixed with 5× sample dye (non-reducing), and electrophoresis separation was performed at 150 V (SDS-PAGE with 2% gelatin in the gel, running buffer: Tris Base, glycine, and 0.1% SDS). After washing the gel with washing buffer (containing Triton X-100, Tris-HCl, CaCl₂, and ZnCl₂), the gel was incubated in a 37 °C incubator for 17–24 h with incubation buffer (containing Triton X-100, Tris-HCl, CaCl₂, and ZnCl₂). The gel was stained with staining buffer (containing methanol, acetic acid, and Coomassie Blue) and persistently destained with destain buffer (containing methanol and acetic acid) until the white band was visualized. The band was analyzed using an Amersham Imager 680 blot and gel imager (GE healthcare) and MultiGauge image analysis software version 3.0 (Fujifilm, Stockholm, Sweden).