Western Blotting and qPCR Analysis of Signaling Proteins

Western blotting was performed as described previously using specific antibodies against RORα, actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), pSTAT3, STAT3, JAK1, JAK2, SRC, PIAS3, and SHP-1 (Cell Signaling Technology, Beverly, MA, USA)^{32 (link)}. Quantitative real-time PCR (qPCR) was performed using an ABI StepOnePlus^TM Real-time PCR system (Applied Biosystems, Foster City, CA, USA) using specific primers (Supplementary Table S1). Relative mRNA levels of target genes were estimated using the equation 2^−ΔCt (ΔCt = Ct of target gene minus Ct of β-actin or 18 S rRNA) and are presented relative to the level of the control group, which was designated as 1. The detailed method for qPCR is described in Han et al.^{32 (link)}.