Anti mr antibody

RNA extraction, reverse transcription and real-time PCR using SYBR green were conducted, as described previously (Wada et al. 2010 (link), Yonezawa et al. 2012) (link). The relative expression of objective mRNAs was calculated as a ratio to that of the 18S ribosomal RNA (animal experiments) or hypoxanthine-guanine phosphoribosyltransferase (HRPT; in vitro experiments). Primer sequences are listed in Table 1. Western blotting was conducted as described previously (Wada et al. 2013) (link). For sampling of culture media, proteins in the media were precipitated by mixing 235:3 with MeOH and CHCl 3 and yielded pellets were suspended in Laemmli buffer. Antibodies utilized for immunoblotting were described below. Anti-NFkB p65, anti-Caspase 1 and anti-IL1b antibodies were purchased from Santa Cruz Biotechnology. Anti-phospho-Ser536-specific NFkB p65 and anti-beta actin antibodies were purchased from Cell Signaling Technology. Anti-MR antibody was from Abcam Japan (Tokyo, Japan). A densitometric analysis of the blotted membrane was conducted using an LAS-4000 Immunoimage analyzer system (Fujifilm, Tokyo, Japan). For the analysis of mature IL1b secretion from adipose tissue, minced eWAT was incubated in serum-free DMEM for 24 h, and IL1b secretion in the culture media from 1 g of eWAT was determined by ELISA (R&D Systems), as described previously (Honda et al. 2014) (link).