Anti trb3

The western blot assay was performed according to the protocol previously established^{15 (link)}. Briefly, the cells were lysed in 0.2% NP-40 lysis buffer (Life technology), followed by the measurement of protein concentration using a bicinchoninic acid protein assay (Thermo Scientific, IL). The protein lysates of cells were then separated by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), blotted onto Immobilon polyvinyl difluoride (PVDF) membrane (Millipore, Billerica, MA), and incubated in the primary antibodies containing anti-Trb3 (Santa Cruz), anti-PPARγ (Santa Cruz), anti-Smurf1, anti-pSmad1/5/8 (Santa Cruz), anti-NF-kB p65 (phospho) or anti-GAPDH (Santa Cruz). The membranes were further incubated in Horseradish Peroxidase (HRP)-conjugated secondary antibody (Millipore, MA), subsequently visualized with chemiluminescent HRP (Denville Scientific, NJ).

Tissues were homogenized in ice-cold lysis buffer (50 mM Tris HCl, pH 7.5, 0.5% Nonidet P-40, 150 mM NaCl, 2 mM EGTA, 1 mM Na₃VO₄, 100 mM NaF, 10 mM Na₄P₂O₇, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin)^{11 (link)}. Tissue extracts were then immunoblotted with the following primary antibodies: anti-p-eIF2α, anti-EIF2α, anti-GCN2, anti-p-HSL, anti-HSL, anti-IRE1α, and anti-p-PKA substrates (1:1000, Cell Signaling Technology, MA, USA); anti-UCP1 and anti-ATF6 (1:1000, Abcam, Cambridge, UK); anti-TH (1:1000, Merck Millipore, Frankfurter, GER); anti-ATF4 and anti-TRB3 (1:500, Santa Cruz Biotechnology, CA, USA), anti-p-GCN2 (1:500, Biorbyt, Cambridge, UK); anti-CHOP, anti-ATF4, anti-PERK, and anti-XBP1s (1:1000, Proteintech, Hubei, P.R.C.); anti-p-IRE1α (1:1000, Epitomics, CA, USA); anti-p-PERK (1:1000, Signalway Antibody, MD, USA); anti-BIP and anti-β-actin (1:2000, Sigma, MO, USA).