Ab31390

Protein extraction was performed on ice, and the reaction solution was pre-cooled with ice. The tissues or cells were washed with cold phosphate-buffered saline (PBS), lysed with radioimmunoprecipitation assay (RIPA) (Beyotime Institute of Biotechnology) lysis buffer, centrifuged (at 10,000 x g for 15 min at 4̊C), discarded, and resuspended in a cell lysate containing phosphatase inhibitor or protease inhibitor (Beyotime, Shanghai, China). Bicinchoninic acid (BCA) protein concentration assay kit (Beyotime Institute of Biotechnology) was used for the determination of protein quality. Then, the protein was added to the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto the polyvinylidene fluoride (PVDF) membranes (EMD Millipore). After 5% milk blocking, membranes were coated with primary antibodies against: Collagen II (ab34712), ATF4 (ab31390), CHOP (ab240220), cleaved caspase-9 (ab2324), and β-actin (as loading control, ab179467) overnight at 4˚C. All the antibodies were purchased from Abcam. Membranes were then incubated with secondary antibody for 1 h at room temperature. Chemiluminescent ECL substrate (Beyotime Institute of Biotechnology) was used to expose the band.

Immunofluorescence on tibial cryosections was essentially performed as previously described [30 ]. The primary antibodies used were anti-COL2A1 (Abcam, ab34712, 1:100), anti-COL10A1 (from Kathryn Song Eng Cheah, University of Hong Kong, 1:500), anti-COL9A1 (Bioworld Technology, BS6945, 1:50), anti-COL11A1 antibody (Abcam, ab64883, 1:100), anti-ATF6 antibody (Abcam, ab37149, 1:100), anti-ATF4 antibody (Abcam, ab31390, 1:100), anti-GRP78 BiP antibody (Abcam, ab21685, 1:100), anti-XBP1 antibody (Abcam, ab37152, 1:100), anti-phospho- Erk1/2 antibody (Cell Signaling Technology, 4370, 1:200), anti-phospho-Stat1 antibody (Cell Signaling Technology, 9167, 1:200). Alexa Fluor 594 conjugate of Concanavalin A (Invitrogen) was used to colocalized with collagens. For ColII/IX/XI antigen retrieval, sections were treated with 2 mg/ml pepsin (Sigma Aldrich) for 10 min at 37 °C. For ColX antigen retrieval, sections were digested with 2 mg/ml hyaluronidase (Sigma Aldrich) for 20 min at 37 °C. The primary antibodies were detected with appropriate Alexa Fluor-conjugated secondary antibodies (Abcam). All sections were mounted with ProLong Gold Antifade Mountant with DAPI (Invitrogen) and visualized using a fluorescent microscope (Zeiss).