Pxi imager

After treatments, OA chondrocytes were harvested, washed with cold PBS and lysed in ice-cold protein lysis buffer (50 mM Tris pH 7.6, 400 mM NaCl, 0.5% NP-40, 1 mM PMSF and 1 × protease inhibitor cocktail (Roche) as described previously (29 (link), 30 (link)). Lysates were clarified by centrifugation (15000g for 15 min at 4°C) and the supernatant was analyzed immediately or stored at −80°C. Equivalent amounts of lysate protein (25 µg) were resolved by 10% SDS–PAGE and transferred to a PVDF membrane (Hybond P, Amersham Biosciences, Piscataway, New Jersey) and the blots were incubated with diluted anti-IL-6 (1:1000), or anti-MCPIP1 (1:1000) or anti-β-Actin (1:5000) primary antibodies in TBST overnight at 4°C. Blots were then incubated with horseradish peroxidase-conjugated secondary antibody and the antibody reactive proteins were visualized and analyzed using the Syngene Pxi imager and the Syngene Image analysis software respectively.

After treatments, OA chondrocytes were harvested, washed with cold PBS and lysed in ice-cold protein lysis buffer (50 mMTris pH 7.6, 400 mMNaCl, 0.5% NP-40, 1 mM PMSF and 1X protease inhibitor cocktail (Roche) as described previously[Haseeb et al., 2013 (link)]. Lysates were centrifuged (15000g for 15 min at 4°C) and the supernatant was used for estimation of protein. Equivalent amounts of lysate protein (20µg) were resolved by 12% SDS–PAGE and transferred to a PVDF membrane (Bio-Rad, USA) and the blots were incubated with diluted anti-LC3 (1:1000), or anti-Beclin-1 (1:1000) or anti-β-Actin (1:2000) primary antibodies in TBST overnight at 4°C. Blots were then incubated with horse radish peroxidase-conjugated secondary antibody and the antibody reactive proteins were visualized and analyzed using the Syngene Pxi imager and the Syngene Image analysis software respectively.

After desired treatment chondrocytes were harvested, washed with PBS and lysed in ice-cold protein lysis buffer (50 mM Tris pH 7.6, 400 mM NaCl, 0.5% NP-40, 1 mM PMSF and 1x protease inhibitor cocktail (Roche, Mannheim, Germany) [17 (link), 29 (link)]. Lysates were clarified by centrifugation (15000g for 10 min at 4°C) and the supernatant was analyzed immediately or stored at −80°C. Equivalent amounts of proteins were resolved by 10% SDS–PAGE and transferred onto a PVDF membrane (Hybond P, Amersham Biosciences, Piscataway, New Jersey, USA). After blocking in 5% milk, membranes were incubated with anti-IL-6 (1:1000), anti-MCPIP1 (1:1000) or anti-β-Actin (1:5000) primary antibodies overnight at 4°C. To detect acetylated histone, cells were lysed in protein loading buffer (0.313 M Tris pH 6.8, 10%SDS, 40% glycerol, 0.05% bromophenol blue and 2% β-ME). Cells were sonicated three times for 10s each and separated on 12% SDS-PAGE. After blocking membranes were incubated with anti-Ac-H3 (1:1000), anti-Ac-H4 (1:1000), anti-H3 (1:1000) or anti-H4 (1:1000) antibodies overnight at 4°C. Every primary antibody incubation was followed by horseradish peroxidase-conjugated secondary antibody (1:5000). Western blots were visualized using Syngene Pxi imager. Quantification of immuno-reactive bands was performed using the Gene Tools Software (Syngene, Frederick, MD, USA).