Polyvinylidene fluoride membranes 0.45 μm

Western blot assay was performed as previously described.²⁴ Briefly, the total protein of glioma tissues was extracted using a protein extraction kit (Beyotime). Equivalent amounts of protein (25‐50 μg) were then electrophoresed and transferred to polyvinylidene fluoride membranes (0.45 μm; Millipore). After being blocked, membranes were incubated with antibodies against ATF6 (24169‐1‐AP, Proteintech), EIF2α (sc‐133132, Santa), p‐EIF2α (# 3398S, CST), p‐IRE1α (sc‐390960, Santa) and GAPDH (60004‐1‐Ig, Proteintech) at 4°C for 16 hours. The membranes were then incubated with appropriate secondary antibodies (ProteinTech). Protein bands of interest were detected and quantified using a chemiluminescence kit (Beyotime), the ChemiDoc™ Touch detection system (Bio‐Rad Laboratories) and Image J software (National Institutes of Health).

MCF-7 cells were exposed to 100 or 200 µM for 24 h. Collected cells and tumour tissues obtained from the two xenografted tumour bearing mice were homogenized in cold radio-immunoprecipitation assay (RIPA) lysis buffer (20–188, Millipore, Billerica, MA, USA) containing 1% cocktail inhibitors (524625, Millipore, Billerica, MA, USA). The protein concentration of cell lysates and tissue lysates were measured by the BCA Protein Assay Kit. Proteins (40 μg) were electrophoresed and separated with 10–12% sodium dodecyl sulphate polyacrylamide gel electrophoresis gel, and then transferred onto a polyvinylidene fluoride membranes (0.45 μm) (Millipore, Billerica, MA, USA), which was blocked with 5% bull serum albumin. After washes, the membranes were incubated with the primary antibodies including Bcl-2, Bax, Bcl-xL, Nrf2, HO-1, SOD1, SOD-2, p-NF-κB, NF-κB, p-IкBα, IкBα, and GAPDH overnight at 4 °C following with exposure to secondary antibody at 25 °C for 2 h. Protein bands was visualized by the Electro Chemi Luminescence (ECL) detection kits (WBULS0500, Merck Millipore, Billerica, MA, USA), and their intensity were quantified with Image J software (NIH, Bethesda, MD, USA).

Cells were solubilized in lysis buffer comprising phosphate-buffered saline solution containing 1× cell lysis buffer (Cell Signaling Technology, Danvers, MA), 1× protease inhibitor cocktail (Roche, Indianapolis, IN), and 1× phosphatase inhibitor cocktail (Roche) and incubated for 30 min on ice. The lysates were then subjected to centrifugation for 10 min at 13,000 rpm at 4°C. Total protein concentrations were determined by the Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's instructions. Total proteins (40 μg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Bio-Rad Laboratories) and transferred to polyvinylidene-fluoride membranes (0.45 μm, Millipore, Bedford, MA), then probed with first and second antibodies according to the manufacturers’ protocols. The following antibodies were used: α-tubulin (Sigma-Aldrich, St Louis, MO), p-HSF1^Ser326 (Abcam, Cambridge, MA), HSF1, p-S6 ribosomal protein (S6K) ^{Ser235/Ser236}, S6 ribosomal protein, c-Myc, cleaved caspase-9, p-AMPKa^Thr172, AMPKα, p-TSC2^Thr1462, TSC2 and horseradish peroxidase–linked anti-mouse and anti-rabbit IgG (all, Cell Signaling Technology).