Protein Extraction and Western Blot Analysis

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).