Anti asic1a

BMSCs were washed with PBS and fixed in 4% paraformaldehyde for 15 min at room temperature. The cells were washed with PBS three times and permeabilized for 15 min in 0.2% Triton X-100 solution. The cells were incubated in 5% bovine serum albumin (BSA) for 60 min to block nonspecific binding. Then, the cells were incubated overnight at 4°C with rabbit polyclonal anti-Ki67 (1:400, Abcam, U.K.), anti-CD29 (1:200, Abcam, U.K.), anti-CD90 (1:200, Abcam, U.K.), anti-CD44 (1:200, Abcam, U.K.), and anti-ASIC1a (1:200, Alomone Labs, Israel) antibodies. Next, the cells were incubated in the dark for 2 h with Alexa Fluor 488- or Alexa Fluor 594-conjugated donkey anti-rabbit IgG at room temperature. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) at room temperature for 2 min. The fluorescence was visualized using a fluorescence microscope (FV2000, Olympus, Japan). Negative control was performed following the same procedure using nonspecific rabbit IgG instead of primary antibodies.

After treatment as indicated earlier, the articular chondrocytes were fixed with ice-acetone for 15 min, permeabilized with 0.3% Triton X-100 in PBS for 15 min, and then blocked with PBS containing 5% bovine serum albumin (BSA, Sigma-Aldrich) for 1 h. The cells were then incubated with anti-ASIC1a (1:100, Alomone Labs, Jerusalem, Israel), anti-FoxO3a (1:100; Cell Signaling Technology, Danvers, MA, USA) antibody overnight at 4 °C, followed by detection with an fluorescein isothiocyanate (FITC)-conjugated anti-rat IgG (Molecular Probes, Beijing, China) in the dark for 1 h at 37 °C. Control sections were treated in parallel with a non-immune isotype-matched IgG substituting for the primary antibody. Nuclear staining was incubated with 4′,6-diamidino-2-phenylindole, dilactate (DAPI; Invitrogen, Carlsbad, CA, USA). Cells were washed and imaged using an inverted fluorescence microscope (Olympus, Tokyo, Japan).

Cells were collected and lysed by strong RIPA lysate containing protease inhibitors (1: 50 dilution, Bioss Biotechnology Company, Beijing, China). BCA‐200 was used to determine the concentration of protein in the lysate. The membranes were sealed with 5% skim milk for 1 h then incubated with anti‐ASIC1a (1: 200; Alomone Laboratories, Jerusalem, Israel), anti‐AKT, Phospho‐AKT, GSK3β, Phospho‐GSK3β, Snail, Smad2/3, Phospho‐Smad2/3, TGF‐β1, β‐actin (1:1000, Cell Signalling Technology, Danvers, MA, USA), anti‐MMP2, MMP9, α‐catenin, β‐catenin, E‐cadherin, vimentin and fibronectin (1:500, Abcam, Cambridge, UK) antibodies overnight at 4°C. The membranes were incubated in TBST buffer solution containing the secondary antibody (1:4000) at room temperature for 1 h. The results displayed by the gel imaging system were compared with the percentage of the control signal to correct for the difference between the imprints.