Pim 1

The knee joints were obtained from the OA and processed with safranin-O and fast green (S&F) and hematoxylin & eosin (H&E). For the OA model, the severity of synovitis was assessed by the synovitis scores and the severity of cartilage damage was calculated by Osteoarthritis Research Society International (OARSI) scoring system [40 (link)]. Immunohistochemical staining was accomplished with the knee joint cartilages. The primary antibodies used in the experiments were those against matrix metallopeptidase 13 (MMP13) (ABclonal), A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) (ABclonal), Collagen II (ProteinTech), Aggrecan (ProteinTech), Cleaved Caspase-3 (Cell Signaling Technology), PIM-1(HUABIO). The images of immunohistochemical staining were captured using an Olympus VS200 system (Tokyo, Japan).

The total proteins from different macrophages and chondrocytes were extracted with the RIPA lysis buffer (MCE, NJ, USA) containing a mixture of phosphatase and protease inhibitors. Then, the proteins were separated with 8–12% SDS-PAGE (Febio science, Hangzhou, China) and transferred to the polyvinylidene fluoride membranes (Merk Millipore, Darmstadt, Germany). The membranes were blocked with 5%BSA and then incubated with the primary antibodies overnight. The membranes were washed next day with PBST and then incubated with the HRP-conjugated secondary antibodies (Beyotime, Shanghai, China) for 90 min. These results on the membranes were captured and documented by Molecular Imager (Bio-Rad, CA, USA).
The primary antibodies used include those against IL-1β (R&D Systems, MN, USA), NLRP3 (Adipogen, CA, USA), ASC (Adipogen), Caspase-1 (Adipogen), P-jak2 (Abcam, MA, USA), P-Stat3 (Abcam), P-bad (Cell Signaling Technology, MA, USA), Cleaved Caspase-3 (Cell Signaling Technology), Jak2 (ABclonal, Wuhan, China), Stat3 (ABclonal), Bad (ABclonal), Clic1 (ABclonal), Clic4 (ABclonal), ADAMTS5 (ABclonal), MMP13 (ABclonal), Bax (HUABIO, Hangzhou, China), Bcl-2 (HUABIO), Clic5 (HUABIO), PIM-1 (HUABIO), Collagen II (ProteinTech, Wuhan, China), Aggrecan (ProteinTech), β-actin (ProteinTech), GADPH (ProteinTech).