Anti p hsl

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).

Protein samples (20 µg) of mouse liver were applied to 8% to 12% SDS-PAGE for electrophoretic separation and immunoblotting. The following were the primary antibodies used in the Western blot assay: anti-p-HSL (#4126; Cell Signaling Technology, Danvers, MA, USA), anti-HSL (#4107, Cell Signaling Technology), anti-PGC1α (#54481, Abcam, Cambridge, UK), anti-PPARγ (#7273, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-C/EBPα (#8178, Cell Signaling Technology), anti- PLIN2 (#108323, Abcam), anti-PCK1 (#70358, Abcam), anti-G6Pc (#83690, Abcam), anti-IRβ (#3025, Cell Signaling Technology), anti-IRS-1 (#3407, Cell Signaling Technology), anti-PI3K (#4257, Cell Signaling Technology), anti-p-AKT (#9271, Cell Signaling Technology), anti-AKT (#9272, Cell Signaling Technology), anti-CAT (#ER40125, HuaBio, Hangzhou, China), anti-SOD2 (#ET1701-54, HuaBio), anti-COX2 (#12282, Cell Signaling Technology), anti-GPX4 (#125066, Abcam), anti-FtH (#81444, Abcam), anti-xCT (#37185, Abcam), and anti-β-actin (#8457, Cell Signaling Technology). Peroxidase-conjugated goat anti-rabbit IgG (BL003A, Biosharp, Hefei, China) and goat anti-mouse IgG (BL001A, Biosharp) were applied as secondary antibodies.

Cells or minced mouse tissue were lysed with 1 x RIPA buffer supplemented with the Halt Protease and Phosphatase inhibitor cocktail (ThermoFisher) for 30 min. Following, protein concentration was quantified, and lysates were then boiled at 95 C in 1 x Laemmli (Bio-Rad) for 5 min. The proteins were separated by SDS-PAGE on 4–15% gradient gels (Bio-Rad), and transferred onto nitrocellulose membranes (Bio-Rad). After 1 hr of blocking in 3% skim milk, membranes were incubated overnight with the following primary antibodies: anti- actin (Santa Cruz), anti-Dnmt3a (Cell signaling), anti-p-HSL (Cell Signaling), anti-STAT3 (Cell Signaling), anti-p-STAT3 (Cell Signaling). This was followed by secondary antibody incubation with anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibody (Santa Cruz), and imaging on the Bio-Rad ChemiDoc platform. Antibodies used: β-Actin Antibody (Cell Signaling, Cat$4,967 S), phosphor-HSL(Ser660) (Cell Signaling. Cat #4,126 S).