Actin antibody ab3280

Anti-TFEB antibody (A303-673A, 1:1000 dilution) was purchased from Bethyl Laboratoryies, Inc. (Montgomery, TX). Actin antibody (ab3280, 1:10,000 dilution), and CBS antibody (ab135626, 1:2000 dilution), and CDO1 antibody (ab53436, 1:2000 dilution) was purchased from Abcam (Cambridge, MA). LC3 antibody (Cat #. 3868 S, 1:2000 dilution) was purchased from Cell Signaling Technology (Danvers, MA). P62 antibody (anti-SQSTM1, #89-015-843, 1:2000 dilution) was purchased from Fisher Scientific (Waltham, MA). MAT1A antibody (#712035, 1: 2000 dilution) and GNMT antibody (PA5-76962, 1: 2000 dilution) were purchased from ThermoFisher Scientific (Waltham, MA). Antibodies against GCLC (1:2000 dilution) and GCLM (1:2000 dilution) were provided by Terrrance J Kavanagh (University of Washington, Seattle, Washington, USA). Chloroquine was purchased from Sigma (St. Louis, MO). AST assay kit, total cholesterol assay kit and TG assay kit were purchased from Pointe Scientific (Canton. MI).

Cytosolic liver extracts were prepared by homogenizing fresh tissue in 10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl₂, 0.5 mM DTT, 0.1% Nonidet-P40, protease inhibitor cocktail and PhosSTOP^TM (all Sigma-Aldrich), leaving it on ice for 30 min with occasional vortexing before centrifuging at 4000×g and 4 °C for 5 min. Supernatant protein concentrations were determined with the BCA assay (Thermo Fisher Scientific, Schwerte, Germany). The samples were mixed with loading buffer, denatured at 95 °C for 5 min and separated on TGX Stain-Free Precast gradient gels (Biorad, Munich, Germany) and blotted onto a PVDF membrane. The membrane was blocked with 5% skim milk dissolved in TBS with 0.05% Tween-20 and probed with a primary antibody overnight (MUP antibody sc-21856, Santa Cruz Biotechnology Inc., Heidelberg, Germany; p-AMPK antibody CS-2523, Cell Signalling, Leiden, The Netherlands; Actin antibody ab3280, Abcam, Cambridge, UK) followed by a secondary antibody (anti-goat sc-2354: Santa Cruz Biotechnology Inc., Heidelberg, Germany, anti-mouse 170–5047; anti-rabbit 170–5046: Biorad). The bands were visualized with ECL reagent (Thermo Fisher Scientific) in a ChemiDoc XRS system (BioRad).