Ampkγ1

Phospho-AMPKα (1:1000, Thr 172) (2535), AMPKα (1:1000, 2603), AMPKα1 (1:1000, 2795), AMPKα2 (1:1000, 2757), AMPKβ1 (1:1000, 4178), AMPKγ1 (1:1000, 4187), phospho-ACC (1:1000, 3661), ACC (1:1000, 3676), phospho-AKT (1:1000, Ser 473) (9271) and AKT1 (1:2000, 2967) antibodies were acquired from Cell Signaling Technology (Danvers, MA, USA). The UCP1 antibody (1:1000, ab10983) and mouse AMPKα1 (1:1000, ab3759) were procured from Abcam (Cambridge, MA, USA). Mouse AMPKα2 (1:1000, AF2850) was purchased from R&D systems Inc. (MN, USA). FLAG (1:3000, F3165 and F7425) and β-actin (1:10000, A5316) antibodies were procured from Sigma-Aldrich. The MKRN1 antibody (1:3000, A300-990A) was procured from Bethyl Laboratories. The mono- and polyubiquitin chain antibodies (1:1000, FK2, Biomol, PW0150) were purchased from Enzo Life Sciences. The HA antibody (1:3000, 12013819001) was obtained from Roche. GAPDH (1:5000, sc-25778), GFP (1:5000, sc-8334) and HA (hemagglutinin) (1:3000, sc-7392 and sc-805) antibodies were procured from Santa Cruz Biotechnology (Dallas, TX, USA). MG132 (M-1157) was purchased from A.G. Scientific (CA, USA). CHX (C4859) was purchased from Sigma-Aldrich.

Cells were grown to 70% confluency on poly-lysine coated cover slips. After treatment, immunofluorescent staining was performed following published procedures [5] (link). Antibody dilutions were: AMPK-α1 and AMPK-α2 (1:1000; Upstate 07-350; 07-363; or 1:2000; Bethyl A300-507A, A300-508A), AMPK-β1/2 (1:400; Cell Signaling ♯4150), AMPK-γ1 (1:200; Cell Signaling ♯4187), AMPK-γ2 (1:200; Cell Signaling ♯2536), HuR (1:2000; sc-5261), G3BP1 (1:1000; BD Biosciences ♯611126). In brief, cells were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS), permeabilized with 0.1% Triton X-100 in PBS/2 mg/BSA and blocked for 1 h in PBS containing 5% fetal bovine serum (FBS), 0.05% Tween-20 and 1 mM NaN₃. Samples were incubated overnight with primary antibodies diluted in PBS/FBS/Tween. Purified Alexa Fluor® 488, Alexa Fluor® 647 and Cy3 fluorescently-labeled secondary antibodies were added the following day for 2 h [6] (link). Nuclei were visualized with 1 µg/ml 4׳,6 Diamidino-2-phenylindole dihydrochloride (DAPI).

Whole-cell lysates were resolved by SDS-PAGE gel electrophoresis, electrotransferred to a PVDF membrane, and probed with indicated antibodies. The following antibodies were purchased from Cell Signaling Technology: AMPKα1 (#2795), AMPKα2 (#2757), Phospho-AMPKα (Thr172) (#2535), AMPKβ1 (#4178), AMPKβ2 (#4148), AMPKγ1 (#4187), AMPKγ2, (#2536), AMPKγ3 (#2550), Acetyl-CoA Carboxylase (#3676), Phospho-Acetyl-CoA Carboxylase (Ser79) (#3661), cleaved Caspase-3 (Asp175) (#9661 and #9664), E-cadherin (#3195), Gasdermin D (#97558 and #93709), and cleaved Gasdermin D (Asp275) (#36425).