Anti phospho eif2α 3398

The western blot procedure was as outlined in our previous research. In short, both HOS cells and tumors were lysed using chilled RIPA buffer (Beyotime Biotech), combined with a phosphatase and protease inhibitor cocktail (Bioss). After separation by 10% or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, the proteins were transferred onto polyvinylidene fluoride membranes (Beyotime Biotech), which were then blocked with 5% non-fat milk for 2 h. The membranes were incubated with the following primary antibodies for a duration exceeding 12 h at a temperature of 4°C: anti-PERK (5683), anti-CHOP (2895), anti-BiP (3177), anti-ATF4 (11815), anti-LC3B (3868), anti-SQSTM1/p62 (88588), anti-Atg5 (12994) anti-cleaved caspase-3 (9664), anti-cleaved PARP (5625), anti-p21 (2947) all at 1:1000, and anti-phospho-eIF2α (3398) at 1:800, all from Cell Signaling Technology. Anti-β-actin (bs-0061R) at 1:5000, anti-phospho-PERK (bs-3330R) at 1:800, and anti-GAPDH (bs-0755R) at 1:5000 from Bioss were also used. Then, the membranes were further incubated with the corresponding HRP-labeled secondary antibodies. Detection was carried out using BiossECL Plus WB Substrate (Bioss), and band intensities were analyzed using Image Lab software.

For Western blotting, 20 μg proteins were applied to lanes of 10% or 4% to 12% Bis-Tris Gels, and then transferred onto Immobilon-P membranes (Millipore, Bedford, MA, USA). The membranes were incubated with the relevant antibodies: anti-PKR (product number: 3210), anti-EIF2α (5324), anti-phospho EIF2α (3398), anti-c-Jun (9165), anti-phospho c-Jun (3270), anti-c-Fos (2250), anti-phospho c-Fos (5348), anti-JNK (9252), anti-phospho JNK (4668), anti-ERK1/2 (4659), anti-phospho ERK1/2 (4370) (Cell Signaling, Danvers, MA, USA), and phospho PKR (44-668G) (Thermo Fisher Scientific), overnight at 4°C. Appropriate species-specific conjugated secondary antibody kits were commercially obtained (GE Healthcare, Charles Coffin, NY, USA). Signals were detected using the ECL Prime Kit (GE Healthcare) with an ImageQuant LAS 4000 system (GE Healthcare).