Anti p ampk antibody

HEK293T cells were lysed by cell lysis solution containing protease inhibitor and phosphatase inhibitor at 4 degrees for 2 h. Cell lysates were then centrifuged to remove cell debris and were treated the sample with β-mercaptoethanol for 30 min, and boiled for 5 min to denature protein. Cell lysates were subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis and incubated with anti-AMPK antibody (Cell Signaling) and anti-p-AMPK antibody (Cell Signaling) at 4 degrees overnight and followed by goat anti-rabbit horseradish-peroxidase-labeled secondary antibody (Santa Cruz) at room temperature for 1 h. The total amounts of pAMPK and total AMPK were measured as described [60 (link),61 (link)], and their relative abundances were quantified using NIH ImageJ software (NIH, Bethesda, MD, USA). All experiments were performed at least three times and representative results were presented. Metformin and DMSO were used as positive controls and vehicle controls, respectively.

Total proteins were extracted as previously described.²³, ²⁶, ²⁷ The tissues were briefly homogenized in radioimmunoprecipitation assay (RIPA) lysis buffer (Biotime) supplemented with 1% phenylmethylsulfonyl fluoride (Sangon Biotech) and 1× PhosSTOP phosphatase inhibitor cocktail (Roche Applied Science). Protein concentration was measured using a butyleyanoacrylate (BCA) kit according to the manufacturer's instructions (Thermo Scientific). The proteins were separated using SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore). The membranes were blocked in 5% bovine albumin (BSA) with a TBST buffer (20 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20), followed by incubation with primary antibodies diluted in TBST containing 2.5% BSA at 4°C overnight: anti‐PPM1F (1:500, PA5‐15571, Invitrogen), anti‐p‐AMPK antibody (1:1000, #2535, Cell Signaling Technology), anti‐AMPK antibody (1:1000, ab32047, Abcam), and anti‐β‐actin antibody (1:1000, 4970, Cell Signaling Technology). Then, the membranes were washed with 1× TBST and incubated with IRDye 680LT donkey anti‐rabbit IgG secondary antibodies (1:5000, 926–68,023, Li‐COR Biosciences). Fluorescence was visualized and analyzed using an Odyssey infrared imaging system (Li‐COR Biosciences).