Ab93357

The joint and lung tissue lysates (20 μg per sample) were resolved on NuPage 4–12% Bis-Tris protein gels (Invitrogen) and transferred onto nitrocellulose membranes. The membranes were blocked overnight with 5% milk powder (w/v) and probed with antibodies for murine CRAMP (rabbit polyclonal, Abcam, USA, catalog number ab93357), S100A8 (rat monoclonal [clone ABM4A69], Abcam, catalog number ab220174), S100A9 (rat monoclonal [clone 2B10], Abcam, catalog number ab105472), α-defensin 1 (goat polyclonal, Abcam, catalog number ab122884), β-defenisn 2 (rabbit polyclonal, MyBioSource, USA, catalog number MBS2005685) and β-defensin 14 (rabbit polyclonal, MyBioSource, catalog number MBS1490249). Antibody to β-actin (Cell Signaling Technologies) was used to normalize for protein loading. Affinity-purified horseradish peroxidase (HRP)-linked secondary antibodies (Cell Signaling, USA) along with Amersham ECL Prime (GE Healthcare) was used for detection. The blots were imaged using AmershamTM Imager 680 blot and gel imager. Densitometry assessment of band intensity was determined using AmershamTM Imager 680 analysis software version 2.0. The relative band intensity was assessed after normalization with the band intensity for β-actin.

Whole cell lysates were extracted in lysis buffer (50 mM Tris HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate) containing cocktails of protease and phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA, USA; 78444). Protein concentrations were determined by BCA assay (Sigma, St. Louis, MO, USA). Culture supernatants were also collected to determine the levels of extracellular Mmp3 and Mmp13. The lysates or supernatants were resolved by SDS-PAGE, transferred onto nitrocellulose membranes, and incubated with antibodies against Cramp (1:1000; Abcam, Cambridge, UK; ab93357), Mmp3 (1:10,000; Abcam; ab52915), Mmp13 (1:500; Aviva Systems Biology, San Diego, CA, USA; ARP56350_P050), Sox9 (1:1000; Abcam; ab185966), or β-actin (1:10,000; Abcam; ab8226).