Westernbright sirius chemiluminescent reagents

Protein from adipose tissue was extracted as previously described ^{25 (link)} and mouse liver nuclear extracts was prepared as in ^{20 (link)}. Protein extract concentration was determined using Bio-Rad protein assay. Proteins were separated on a SDS-PAGE and electro-transferred to a polyvinylidene fluoride membrane. The membrane was incubated in 5 % skim milk in 50 mM Tris-HCl (pH 7.5), 0.1M NaCl, 0.05 % Tween-20 for 1hr at room temperature. Phosphorylated RXRα was detected with anti pThr 167 RXRα antibody diluted in 5% BSA by incubating overnight at 4 °C. Washed membrane was incubated with secondary antibody (HRP-conjugated anti rabbit IgG) and positive bands were visualized with WesternBright Sirius chemiluminescent reagents (Advansta) and recorded with a western blot scanner (Licor). For detecting transfected flag-tagged RXRα in cultured cell line, anti-Flag antibody ²⁶ .

Liver from PXR WT or PXR KI mice was homogenized using a Dounce homogenizer in T-PER buffer (Thermo Fisher Scientific). The proteins were separated by SDS-PAGE and transferred onto the PVDF membrane. The membrane was incubated with an anti-PXR antibody (#39589, Active motif, Carlsbad, CA, USA). Protein bands were visualized with WesternBright Sirius chemiluminescent reagents (Advansta, San Jose, CA, USA) and recorded with a western blot scanner (Licor, Lincoln, NE, USA).