Irdye 680lt anti mouse

Primary Abs used for immunoblotting were anti-β-actin (AC-74) and anti-Flag (M2) from Sigma, anti-IκBα (a gift from Prof. R. Hay, Dundee University, UK), anti-phospho-JNK (Thr183/Tyr185, 44-682G) from Biosource, anti-p38 (#9212), anti-phospho-p38 (Thr180/Tyr182, #9211), anti-JNK (#9252), anti-phospho-ERK1/2 (Thr202/Tyr204, #4377) and anti-phospho-STAT1 (Tyr701, #9171) from Cell Signaling Technology. The secondary Abs for immunoblotting were IRDye 680LT anti-mouse, IRDye 800CW anti-rabbit and IRDye 680LT anti-goat (LI-COR Biosciences). Primary Abs for confocal microscopy were anti-p65 (F-6, sc-8008) from Santa Cruz and anti-IRF3 (#51-3200) from Invitrogen, while secondary Abs were Alexa Fluor 647 anti-mouse or Alexa Fluor 488 anti-rabbit (Invitrogen). Abs used for ChIP were anti-Pol II (N-20, sc-899X), anti-p65 (C-20, sc-372X), anti-IRF1 (M-20, sc-640X) and anti-IRF8 (C-19, sc-6058X) from Santa Cruz, anti-IRF3 (#51-3200) from Invitrogen and isotype-control rabbit IgG (Sigma). The neutralizing IFNAR1 Ab (MAR1-5A3, #16-5945) was purchased from eBioscience.

Forty nanograms of protein extracted from frozen adrenals tissues in RIPA buffer (50 mM Tris- HCl, pH7.4, 150 mM NaCl, 1% Triton X-100, 0.5% Na-deoxycholate, 1mM EDTA, 10 mM NaF, protease and phosphatase inhibitors (EMD Biosciences)), was separated in 10% SDS Page and transferred onto nitrocellulose membrane. Proteins were then incubated with anti-rabbit anti-P450 aromatase (ab18995, Abcam) and anti-mouse anti-GAPDH (sc-47724, Santa Cruz) primary antibodies overnight at 4C before recognition by the IRDye 800CW anti-rabbit (926–68073, LiCOR) and IRDye 680LT anti-mouse (926–32212, LiCOR) secondary antibody, respectively. Signals is detected by LiCOR CLx and CYP19 expression was quantified using Image StudioLite.