Immunoblot Analysis of Protein Expression

Immunoblot analysis was performed as previously reported^{12 (link)}. Total proteins were extracted from ciBAs and the control fibroblasts with RIPA buffer [50 mM Tris–HCl (pH 8.0), 0.15 M sodium chloride, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 1% NP-40 substitute; Wako] and protease inhibitor cocktail (Wako). The proteins were subjected to 10% SDS-PAGE and transferred to a PVDF membrane (Thermo Fisher Scientific, DE, USA). The membranes were blocked with 5% skim milk followed by incubation with UCP1 antibody (MAB6158, R&D Systems, MN, USA) or β-Actin antibody (A5316, Sigma-Aldrich, MO, USA) at 4 °C overnight. Total SMAD2/3 proteins and phosphorylated SMAD2/3 proteins were detected by Smad2/3 (D7G7) XP Rabbit mAb (#8685, Cell Signalling Technology, MA, USA) and Phospho-Smad2 (Ser465/567)/Smad3 (Ser423/425) (D27F4) Rabbit mAb (#8828, Cell Signalling Technology), respectively. The membranes were incubated with either HRP-conjugated anti-rabbit or anti-mouse secondary antibody (Santa Cruz Biotechnology, CA, USA) for 1 hr at room temperature. Immunoreactive bands were detected by Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, Darmstadt, Germany). Each band intensity was quantified by densitometry using ImageJ software (National Institutes of Health).