Ps108

The protein was extracted from the cells using radio immunoprecipitation assay (PC101, RIPA) lysis buffer (EpiZyme, Shanghai, China). The protein concentration was determined using Thermo Scientific™ Pierce™ BCA (23227). An equal amount of protein was separated by 10–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After blocking with protein-free fast blocking buffer (PS108, EpiZyme), it was incubated with primary antibodies against Runx2 (Affinity, AF5186), Bcl-2 (Affinity, AF6139), Caspase-3 (Huabio, ET1608–64), and GAPDH (EpiZyme, LF205) at 4 °C overnight, and then incubated with the secondary antibody at 37 °C for 1 h. Thereafter, the proteins were visualized using Omni ECL reagent (SQ201, ECL; EpiZyme) under e-Blot (Touch Imager, Shanghai, China). The gray densitometric was analyzed using Image J (USA).

Total protein was extracted from A549 cells and lung tissues using radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China). After the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, they were transferred to membranes, which were blocked with Protein Free Rapid Blocking Buffer (PS108, EpiZyme) and incubated with primary antibodies against CHOP (A5462, Bimake), BiP (11587-1-AP; Proteintech, Wuhan, China), ATF6 (D262665, Sangon, China), ATF4 (A5514, Bimake), XBP-1s (24868-1-AP, Proteintech), XBP-1u (25997-1-AP, Proteintech), IRE1α (A00683-1, Boster, Wuhan, China), phospho-IRE1α (S724, human) (ab124945, Abcam, Cambridge, UK), phospho-IRE1α (S724, mouse) (530878, ZEN-BIO, Chengdu, China), Vimentin (ET1610-39, HuaBio, Hangzhou, China), and E-cadherin (340341, ZEN-BIO) overnight. GAPDH (bs-0755R, Bioss, Beijing, China) was used as the internal reference protein. After incubation with HRP-conjugated goat anti-rabbit IgG (H + L) (AS014, ABclonal, Wuhan, China) and HRP-conjugated goat anti-mouse IgG (H + L) (AS003, ABclonal), the protein bands were visualized using a ChemiDoc™ Imaging System (Bio-Rad) and quantified using NIH ImageJ software (http://rsb.info.nih.gov).

The proteins were extracted and separated using the Epizyme (#PG112) kit before being transferred to a PVDF membrane (Epizyme, #WJ001). For 10 min, a protein-free rapid-blocking solution (Epizyme, #PS108) was used to block the cells. The primary antibody was incubated at 4°C overnight before being rinsed three times for 10 min with Tris-buffered saline containing Tween 20 (TBST). The secondary antibodies were then incubated at room temperature for another 1 h. The Bio-Rad X-ray development system was used to obtain the results after the ECL luminescent substrate (Tanon, #180-501) was added dropwise to the membrane (Bio-Rad, CA, USA). The loading control was -Actin, and the data were analyzed using Image J software. The antibodies used for Western Blot in this study are listed in Supplementary Table 1.

Western blot analysis was performed as described previously^{10 (link)}. Briefly, the total cellular protein was isolated from human and mouse chondrocytes and SW1353 cells using radioimmunoprecipitation assay lysis buffer (Beyotime Biotechnology) containing protease inhibitors (1:100, Abcam, Cambridge, UK). Electrophoresis was started at 80 V for 20 min and then continued using 120 V for 1 h, followed by 250 mA transfer for 1.5 h. After blocking for 15 min with protein-free rapid blocking buffer (PS108, Epizyme, Shanghai, China), the membranes were incubated overnight at 4 °C with primary antibodies (Supplementary Table 2). Following incubation, the membranes were treated with the corresponding horseradish peroxidase-conjugated secondary antibodies (1:3000, Cell Signaling Technology, Danvers, MA, USA) at 20–25 °C for 1 h. The protein bands were detected using a ChemiDoc Touch (Bio-Rad Laboratories, Hercules, CA, USA) and analyzed using Image Lab^TM (Bio-Rad Laboratories). The intensity of bands was compared using ImageJ software (NIH, Bethesda, MD, USA).