Chx c4859

Phospho-AMPKα (1:1000, Thr 172) (2535), AMPKα (1:1000, 2603), AMPKα1 (1:1000, 2795), AMPKα2 (1:1000, 2757), AMPKβ1 (1:1000, 4178), AMPKγ1 (1:1000, 4187), phospho-ACC (1:1000, 3661), ACC (1:1000, 3676), phospho-AKT (1:1000, Ser 473) (9271) and AKT1 (1:2000, 2967) antibodies were acquired from Cell Signaling Technology (Danvers, MA, USA). The UCP1 antibody (1:1000, ab10983) and mouse AMPKα1 (1:1000, ab3759) were procured from Abcam (Cambridge, MA, USA). Mouse AMPKα2 (1:1000, AF2850) was purchased from R&D systems Inc. (MN, USA). FLAG (1:3000, F3165 and F7425) and β-actin (1:10000, A5316) antibodies were procured from Sigma-Aldrich. The MKRN1 antibody (1:3000, A300-990A) was procured from Bethyl Laboratories. The mono- and polyubiquitin chain antibodies (1:1000, FK2, Biomol, PW0150) were purchased from Enzo Life Sciences. The HA antibody (1:3000, 12013819001) was obtained from Roche. GAPDH (1:5000, sc-25778), GFP (1:5000, sc-8334) and HA (hemagglutinin) (1:3000, sc-7392 and sc-805) antibodies were procured from Santa Cruz Biotechnology (Dallas, TX, USA). MG132 (M-1157) was purchased from A.G. Scientific (CA, USA). CHX (C4859) was purchased from Sigma-Aldrich.

Human HCC cells (Hep3B, and HepG2, SMMC7721, MHCC97H, HCCLM3 and Huh-7) and HEK293T cell lines were purchased from the American Type Culture. And these cell lines were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. All cell cultures were maintained at 37°C in a humidified incubator with 5% CO₂. Anti-RNF2 (ab3832 and ab28629) antibodies were purchased from Abcam. Anti-FLAG, anti-HA, anti-Lamin, anti-Tubulin, and anti-Actin antibodies were from Sigma. CHX (C4859), and puromycin (P8833) were from Sigma. MG132 was from Calbiochem. Transfections were performed in 35×10–mm tissue culture dishes by using 1.0 μg of DNA and 2.5 μl of Lipofectamine 2000 (Invitrogen). Immunofluorescence or immunoprecipitation experiments were performed 24 hours later.

Mouse monoclonal anti-Myc-Tag (2276), rabbit monoclonal anti-Myc-Tag antibody (2278), anti-β-TRCP (4394), anti-Cyclin D1 (2978), anti-LRP6 (3395), rabbit polyclonal anti-Cullin 1 (4995) antibodies were purchased from Cell Signaling. Anti-HA antibody (SC-805), anti-Tubulin (SC-73242), anti-c-Myc (SC-40), anti-Axin-2 (SC-8570) and anti-p27 (SC-527) antibodies were purchased from Santa Cruz. Anti-ZNRF3 antibody (ab176449) was purchased from Abcam. Anti-GFP (8371-2) antibody was purchased from Clontech. Anti-β-catenin (AV100600), polyclonal anti-Flag antibody (F7425), monoclonal anti-Flag antibody (F-3165, clone M2), anti-Vinculin antibody (V-4505), peroxidase-conjugated anti-mouse secondary antibody (A-4416), peroxidase-conjugated anti-rabbit secondary antibody (A-4914), anti-HA agarose beads (A-2095) and anti-Flag agarose beads (A-2220) were purchased from Sigma. All antibodies were used in 1:1000 dilutions in 5% non-fat milk for Western blot. Proteasome inhibitor MG132 (S2619) and CKI inhibitor D4476 were obtained from Selleckchem. CHX (C4859) was purchased from Sigma; the neddylation inhibitor MLN4924 was a kind gift from Dr. William Kaelin (Dana-Farber cancer institute). λ-PPase (P0753S) was obtained from New England Biolabs.