Mouse anti hif 2α

Total cell lysates were prepared in lysis buffer [150 mM NaCl, 1% NP-40, 50 mM Tris, 0.2% sodium dodecyl sulfate (SDS), 5 mM NaF] containing a cocktail of protease inhibitors and phosphatase inhibitors (Roche). The following antibodies were used for Western blotting to detect cellular proteins: rabbit anti-Regnase-1 (Novus Biologicals), rabbit anti-SOX9 (Abcam), mouse anti-HIF-2α (Santa Cruz Biotech), and goat anti-Lamin B (Santa Cruz Biotech). For detection of secreted proteins (MMP3 and MMP13), 900 μl of serum-free conditioned medium was subjected to trichloroacetic acid precipitation, and the proteins were fractionated by SDS-PAGE, transferred to a nitrocellulose membrane, and detected using rabbit anti-MMP3 (Abcam) and rabbit anti-MMP13 (Aviva Systems Biology) antibodies.

Total proteins were extracted with lysis buffer (150 mmol/L NaCl, 1% NP‐40, 50 mmol/L Tris, 0.2% sodium dodecyl sulphate and 5 mmol/L NaF) supplemented with a protease inhibitor and phosphatase inhibitor cocktail (Roche, Madison, WI, USA). MMP3 and MMP13 were detected after trichloroacetic acid precipitation as described previously.23 Each protein was visualized using the SuperSignal West Dura kit (Thermo Scientific, Waltham, MA, USA); total extracellular signal‐regulated kinase (ERK) was used as a loading control. Western blot analysis was performed to detect protein levels using the following antibodies: mouse anti‐Hif‐2α (sc‐13596; Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti‐Mmp3 (ab52915; Abcam, Cambridge, UK), mouse anti‐Mmp13 (ab51072; Abcam), goat anti‐COX‐2 (sc‐1745; Santa Cruz), mouse anti‐Erk1/2 (610408; Becton Dickinson, NJ, USA), mouse anti‐pErk1/2 (9101; Cell Signaling Technology, Boston, MA, USA) and mouse anti‐IκB (9242; Cell Signaling Technology). Anti‐Hif‐2α antibody (ab8365; Abcam) was used for immunostaining as previously described.14

Sagittal Section (5-μm thick) were placed on slides for immunohistochemical staining. The sections were incubated in 3% H₂O₂ for 10 min to block endogenous peroxidase activity and incubated in 0.1% trypsin for 30 min at 37 °C for antigen retrieval. After blocking with 1% BSA for 30 min, the slides were reacted with rabbit anti-HIF-2α (Novus Biologicals), mouse anti-p16 (MA5-17142, Invitrogen, Carlsbad, CA, USA), and mouse anti-p21 (AHZ0422, Invitrogen) antibodies, stained using EnVision+System-HRP (Dako, Denmark) and AEC+ substrate and counterstaining with hematoxylin (Dako). The primary antibodies used for double immunofluorescence staining of mouse tissues included mouse anti-HIF-2α (sc13596; Santa Cruz Biotech., Dallas, TX, USA), rabbit anti-OCN (AB10911; Millipore, Burlington, MA, USA), and rabbit anti-cathepsin K (ab19027; Abcam, Cambridge, MA, USA) antibodies. Proteins were visualized using Alexa 488- or Alexa 594-conjugated secondary antibodies (Thermo Fisher Scientific). Nuclei were visualized with DAPI.