Ampkβ1

ACC (catalog no.: 3676), phospho-ACC1 (Ser79; catalog no.: 3661), AMPKα (catalog no.: 2532), phospho-AMPKα (Thr172; catalog no.: 2535), AMPKβ1 (catalog no.: 4178), Raptor (catalog no.: 2280), phospho-Raptor (S792; catalog no.: 2083), AMPKβ1 (catalog no.: 12063), AMPKβ2 (catalog no.: 4148), and AMPKβ1/2 (catalog no.: 4150) were obtained from Cell Signaling Technology. Antibody against AMPKγ1 (catalog no.: ab32508) was purchased from Abcam, and tubulin (catalog no.: T6074), FLAG (catalog no.: F7425), and β-actin (catalog no.: A2228) were purchased from MilliporeSigma. HTRF assay for detection of phospho-ACC was obtained from Cisbio (catalog no.: 64ACCPET).

Primary hepatocytes were isolated from two male C57BL/6 mice using methods described elsewhere (60 (link)) and plated in 6-well collagen I-coated plates at a density of 3 × 10⁵ cells/well in Williams’ E medium containing 10% fetal bovine serum. Primary hepatocytes were serum-starved in plain William's E medium for 2 h prior to treatment with a DMSO vehicle control or 4-HPAA (TCI America Product No. H0290) for 30 min. Cell pellets or whole liver homogenates were prepared from primary hepatocytes or murine liver, respectively, in a modified RIPA buffer with protease and phosphatase inhibitors. Western blotting was performed as previously described (61 (link)). Images were captured using a GE Amhersham Imager 6000, and the accompanying software (version 1,1.1) was used for densitometric protein expression analysis. Primary antibodies (pS79-ACC, Cell Signaling, #11818; ACC, Cell Signaling, #3676; pT172-AMPKα, Cell Signaling, #2535; AMPKα, Cell Signaling, #5831; pS182-AMPKβ1, Cell Signaling, #4186; AMPKβ1, Cell Signaling, #4178) were prepared 1:1,000 in TBST buffer with 5% (w/v) BSA. Rabbit secondary (Cell Signaling, #7074) and β-actin (Cell Signaling, #12620) antibodies were prepared 1:5,000 in TBST with 5% (w/v) nonfat dried milk.