Ab52915

Immunofluorescence staining was performed on the sagittal sections of the murine knee joint to estimate the severity of OA. The incubation of primary antibody against MMP13 (rabbit, cat. no. #18165-1-AP; Proteintech) and collagen II (rabbit, cat. no. #ab34712; Abcam) on the slices was administered after antigen retrieval and blocking. Then, the sections were probed with fluorescein isothiocyanate–conjugated secondary antibodies (Alexa Fluor 555-labeled Donkey Anti-Rabbit IgG (H + L) and Alexa Fluor 488-labeled Donkey Anti-Rabbit IgG (H + L), Beyotime Institute of Biotechnology). The nuclei were counterstained with DAPI (Sigma, D9542). The images were viewed using a Nikon A1 confocal microscope and analyzed using ImageJ software.
For immunohistochemistry, the knee slices were prepared as described in the histological analysis and subsequently subjected to antibodies against MMP3 (rabbit, cat. no. #ab52915; Abcam) and ADAMTS5 (rabbit, cat. no. #ab41037; Abcam). Percentages of positive MMP3 and ADAMTS5 chondrocytes were determined by counting the number of immunostained cells and dividing by the total number of chondrocytes visualized by a hematoxylin counterstain.

Cells were lysed on ice for 30 min in lysis buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, and 0.1% SDS supplemented with protease inhibitors (10 mg/ml leupeptin, 10 mg/ml pepstatin A, and 10 mg/ml aprotinin). Protein fractions were collected by centrifugation at 10,000 × g for 10 min, separated on 10% SDS-PAGE gels, and then electrotransferred onto nitrocellulose membranes (Whatman, Piscataway, NJ, USA). The membranes were blocked with 5% BSA and then incubated with specific antibodies overnight at 4 °C. The primary antibodies and their sources were as follows: COL2A1 (1:1000, GB11021, Servicebio), ADAMTS4 (1:1000, ab185722, Abcam), p65 (1:5000, ab32536, Abcam), MMP3 (1:1000, ab52915, Abcam), MMP13 (1:1000, ab84594, Abcam), Caspase-7 (1:1000, DF6441, Affinity), cleaved Caspase-3 (1:1000, AF7022, Affinity), Caspase-3 (1:1000, AF6311, Affinity), Bcl-2 (1:2000, ab182858, Abcam), Bax (1:1000, ab32503, Abcam), TNF alpha (1:1000, ab1793, Abcam), β-actin (1:1000, ab6276, Abcam), and GAPDH (1:2000, AF7021, Affinity). HRP-conjugated secondary antibodies (ab205719 and ab6721, Abcam) were used at a 1:2000 dilution. The antigen–antibody complexes were visualized using an enhanced chemiluminescence detection system (Millipore, Darmstadt, Germany) according to the manufacturer’s instructions.

The protein concentration was determined using a Bicinchoninic Acid (BCA) kit (Pierce, Rockford, IL, United States). The total protein was separated by 10% SDS-PAGE electrophoresis. After 2 h of electrophoresis, the protein was transferred to PVDF membranes (Amersham, Buckinghamshire, United Kingdom), which were sealed with 5% skimmed milk powder for 1 hour and incubated with the Anti-MMP3 antibody (1:1,000, ab52915, Abcam), Anti-MMP9 antibody (1:1,000, ab76003, Abcam), Anti-MMP13 antibody (NBP2-45887, 1:1,000, Novus, Biologicals, CO, United States), Anti-RECK antibody (Cell Signaling Technology, CST, Danvers, MA, United States), Anti-COX2 antibody (1:1,000, ab179800, Abcam), Anti-MIP-1β antibody (1:1,000, ab45690, Abcam), Anti- VCAM-1 antibody (1:1,000, ab134047, Abcam), Anti-ICAM-1 antibody (1:1,000, ab171123, Abcam), Anti-HIF-1α antibody (1:1,000, ab179483, Abcam), Anti-β-actin antibody (1:1,000, ab179467, Abcam), and Anti-GAPDH antibody (1:1,000, ab9485, Abcam) overnight. The following day, the membranes were rinsed with TBST. Horseradish peroxidase-labeled secondary anti-Goat anti-Rabbit IgG (1:3,000, ab150077, Abcam) was added and incubated at 37°C for 1 hour. The images were developed and preserved by ECL (Beyotime, Shanghai, China). The experiment was repeated three times, and each time was performed in triplicate.

Cartilage tissues and primary chondrocytes were lysed to obtain protein extracts, which were subjected to SDS‐PAGE (8%‐12% gel) and then transferred to polyvinylidene fluoride membranes (Millipore: Billerica, MA, USA). Membranes were blocked for 1 hour with 5% non‐fat milk prepared in Tris‐buffered saline (TBS) containing 0.1% Tween 20 (TBST) at room temperature (RT). Next, membranes were incubated overnight at 4°C with specific primary antibodies against PIK3R1 (Proteintech: Chicago, IL, USA, 60225‐1‐I, 1:500), cleaved caspase‐3 (Cell Signaling: Beverly, MA, USA, #9661, 1:1000), collagen Ⅱ (Novus Biologicals: Littleton, CO, USA, NBP1‐77795, 1:2000), aggrecan (abcam, ab3778, 1:1000), MMP3 (abcam: Cambridge, MA, USA, ab52915, 1:2000), MMP13 (abcam, ab39012, 1:2000) and GAPDH (Santa Cruz Biotechnology: Santa Cruz, CA, USA, sc‐32233, 1:5000). After washing with TBST, membranes were incubated with horseradish peroxidase‐labelled secondary antibodies at RT for 1 hour. The signal was visualized using an enhanced chemiluminescence reagent according to the manufacturer's protocol (Millipore: Billerica, MA, USA). GAPDH served as a loading control. The protein expression was quantified by ImageJ (http://rsb.info.nih.gov/ij/).