Hrp conjugated anti mouse antibody

50ng/μl type II collagen were coated with 100 μl coating buffer (BioLegend, San Diego, CA, US) on Corning Costar Brand 96-Well EIA/RIA Plate (Thermo Fisher, Waltham, Massachusetts, US) overnight. After washing and blocking with 1% BSA buffer, mice sera or joint lysate (as mentioned in cytokine assay) were diluted from 1:10 to 1:107 with three times dilution and incubated for 2h at room temperature. After wash, HRP conjugated anti-mouse antibody (abcam, Cambridge, UK, 1/10000 dilution) was added in each well for 1h at room temperature, then TMB substrate (abcam, Cambridge, UK) was added for 15 min and reactions stopped with stop solution (Thermo Fisher, Waltham, Massachusetts, US), the color intensity was analyzed at 450nm, and the antibody titer was determined by the OD value that was 3 times higher than that in naïve mice.

Western immunoblotting was performed following standard protocol. Sf9 cells expressing Ss-CYP450 enzymes were pelleted and lysed in Laemmli sample buffer by sonication followed by 5 min in a boiling water bath and 2 min on ice. The samples were then analyzed by 10% SDS–PAGE transferred to nitrocellulose membrane, and immunoblotted with the mouse anti-HA antibody [HA.C5] (Abcam, Cambridge, MA) followed by HRP-conjugated anti-mouse antibody (Abcam, Cambridge, MA). The membranes were then imaged by ImageQuant LAS4000 luminescent Image Analyzer (GE Healthcare, Chicago, IL) following an incubation with ECL chemiluminescent substrate reagent kit (Thermo Fisher, Waltham, MA).