Fresh cell lysis buffer

Cells were lysed in fresh cell lysis buffer (Cell Signaling Technology, Boston, MA, USA) containing 1 μM PMSF, and HALT protease and phosphatase inhibitors (Thermo Scientific, Carlsbad, CA, USA). Twenty micrograms of protein from each sample was resolved on a 12.5% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. After blocking with 5% blocking reagent (BioRad, Hercules, CA, USA) in Tris-buffered saline with 0.5% Tween-20 (TBS-T), the membrane was incubated overnight at 4 °C with either anti-NF-κB p65 (C22B4), anti-phospho-NF-κB p65 (Ser536), or β-actin (8H10D10) primary antibody (Cell Signaling Technology, Boston, MA, USA), followed by incubation with HRP-conjugated anti-rabbit secondary antibody for 2 h at room temperature. Signals were then detected by exposure to X-ray films after the addition of ECL substrate. Densitometry analysis was done using the ImageJ v1.47 (National Institutes of Health, Bethesda, Md, USA) software.

After drug treatment, PC12 cells were washed twice with PBS, and cell pellets were lysed on ice for 30 min with 50 μL of fresh cell lysis buffer (Cell Signaling Technology, cat. no. 9803). The lysate was ultrasonicated and then centrifuged at 12 000 r (4°C) for 15 min. Protein concentration was measured using a BCA kit and then divided into equal protein amount for boiling and loading. After electrophoresis, protein was transferred to PVDF membrane and incubated in 10% nonfat milk at room temperature for 90 min. Blot membranes were incubated with primary antibodies against GRP 78, GRP 94, IRE1, p-PERK, ATF6, p-GSK-3β, and GSK-3β overnight at 4°C. After incubating the secondary antibody at room temperature for 1 h, ECL fluorescence was developed and the blot image was captured and analyzed with Biospectrum Imaging System (UVP, Upland, USA).