P ampk 2535

RtEGM Retinal Pigment Epithelial Cell Growth Medium (RtEGM BulletKit #195409) was purchased from Lonza (Walkersville, MD) and fetal bovine serum (#10438034) were purchased from Thermo Fisher Scientific (Waltham, MA). CFB antibody (sc-271636) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies for AMPK a1 (Abcam) : 32047,AMPKa2 (Abcam) : 3760, were from Abcam  and (P-ACC (#3661), ACC (#3676), AMPK (#2603), P-AMPK (# 2535), and GAPDH (#2118)  were purchased from Cell Signaling Technologies (Beverly, MA). AICAR (#A611700), a pharmacological activator of AMPK, was purchased from Toronto Research Chemicals (Toronto, ON, Canada). 5-iodotubericidin (IODO #I100), dipyridamole (DPY #D9766) and nicotinamide (N3376) were purchased from Sigma (St. Louis, MO). TNF-α  (#210-TA-020) was purchased from R & D Systems (Minneapolis, MN).

Cells or liver tissues were collected and frozen quickly to avoid degradation or dephopsphorylation. The samples were homogenized in a RIPA lysis buffer including protease and phosphatase inhibitors and incubated on ice for 20 min. The supernatant was collected by centrifuging at 14,000 g at 4 °C for 10 min. Protein samples were separated with SDS-PAGE, and then transferred to Polyvinylidene fluoride membranes (Roche, Mannheim, Germany). The membranes were incubated to the primary antibodies and respective secondary antibodies. Primary antibodies for phosphorylated adenosine monophosphate activated kinase (pAMPK, #2535), AMPK (#2532), phosphorylated acetyl-CoA carboxylase (pACC, #3661), and ACC (#3662) were purchased from Cell Signaling Technology. Primary antibodies for glucose transporter 4 (GLUT4) (ab654) and β-actin (A2228) were purchased from Abcam (Cambridge, UK) and Sigma-Aldrich (Saint Louis, MO, USA), respectively. The binding of all antibodies was detected using an ECL detection system (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. The Multi-Gauge Version 3.0 image analysis program (Fujifilm, Tokyo, Japan) was used to measure band densitometry.