Immunofluorescence Staining of AMPK Subunits

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).