Enhanced chemi luminescence (ecl)

At the scheduled time points after incubation, cellular proteins in samples were extracted by lysing with radioimmunoprecipitation assay (RIPA; Beyotime) buffer. Proteins (20 μg) were resolved by SDS–PAGE (Beyotime) and transferred to nitrocellulose membranes (Beyotime). After closure with 5% skim milk or 5% BSA for at least 1 h, membranes were incubated separately with primary antibodies against β-Actin (1:1000), NFATc1 (1:1000), MMP9 (1:1000), CTSK (1:1000), IκB-α (1:1000), phospho-P38 (1:1000), P38 (1:2500), phospho-JNK (1:5000), JNK (1:2000), phospho-ERK (1:1000), ERK (1:1000) and TRAF6 (1:5000) at 4 °C overnight. The corresponding secondary antibody dilutions (1:1000) were then added for 1 h at room temperature after washing membranes adequately with Tris-buffered saline Tween (TBST) for an appropriate time and frequency. Finally, to show the protein bands, membranes were treated with enhanced chemiluminescence (ECL; Yeasen) reagents and imaged using Image Lab 3.0. Protein quantification (relative grey level of the bands) was measured using ImageJ software.

Total protein was extracted from the cells with radioimmunoprecipitation assay (RIPA) buffer (Beyotime Biotechnology Shanghai, China) supplemented with protease inhibitors (Beyotime) and phosphatase inhibitors (Beyotime). The membranes were then blocked with 5% skim milk for 90 min at room temperature and incubated with the primary antibodies overnight at 4 °C. The blots were then washed 3 times for 10 min with TBST (TBS with 0.1% Tween 20) and incubated with HRP-conjugated goat anti-rabbit or mouse IgG secondary antibodies (1:5000) for 1 h at room temperature. The bands were visualized by incubating the blots with enhanced chemiluminescence (ECL, Yeasen) solution and were imaged by a Bio-Rad Imaging system detector.

Western blots were performed on isolated subcellular fractionations resolved in lysing buffer containing: 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 2 mm EDTA, 2 mm EGTA, and 1% SDS supplemented with a protease inhibitor mixture (NCM Biotech, Suzhou, China). Protein samples were separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted to PVDF membranes (Millipore, Sigma-Aldrich, Ireland). After blocking in 5% non-fat milk, the following primary antibodies were used: anti-Calnexin rabbit pAb (1:1000, Cat.A15631, ABclonal, Wuhan, China), anti-Calreticulin rabbit pAb (1:1000, Cat.A18013, ABclonal), anti-GRP75 mouse mAb (1:1000, Cat. sc-133137, Santa Cruz, CA, USA), anti-IP3R rabbit pAb (1:1000, Cat. ab5804, Abcam, MA, USA), anti-VDAC1 mouse mAb (1:1000, Cat. Ab186321, Abcam), anti-TOMM20 rabbit mAb (1:1000, Cat. Ab187735, Abcam,), anti-SAMM50 rabbit pAb (1:1000, Cat. ab246987, Abcam), anti-MIC60 (1:1000, Cat. 10179-1-AP, Proteintech, Chicago, USA), anti-MTX1 rabbit pAb (1:1000, ab233205, Abcam), anti-GAPDH Rabbit mAb (1:5000, Cat. A19056, ABclonal). After incubation of the blots with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h, signals were detected using the ECL (YEASON, Shanghai, China). Quantification was performed with Image Lab software for the ChemiDoc XRS system (Bio-Rad, Hercules, CA).