Ampk γ2

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).

Rabbit polyclonal antibodies (pAb) AMPKα, pThr172−AMPK, ACC, pSer79−ACC, AMPK γ₂ (1:100 for immunoprecipitation), APP (1:500 for western blot), and a mouse monoclonal antibody (mAb) against Tau phosphorylated at Ser396 (1:1000 for western blot) were purchased from Cell Signal Technology. Rabbit pAb against AGEs (1:100 for immunoprecipitation, 1:200 for immunohistochemistry, 1:500 for western blot and dot blot) and mouse mAb Tau 5 against total tau (1:1000 for western blot) were purchased from Abcam. Rabbit pAb against Tau phosphorylation at Ser404 (1:1000 for western blot) was purchased from SAB. A mAb against β−actin (1:1000 for western blot), MG (M0252), and L−arginine (11009–100G−F) were purchased from Sigma Aldrich. Dulbecco’s modified Eagle’s medium (DMEM), Opti−MEM/Reduced−Serum Medium, and phosphate buffer saline (PBS) were purchased from Gibco. FBS (Certified Fetal Bovine Serum) was from biological industries, 04−001−1ACS.