Ha hemagglutinin

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.

Eukaryotic expression vectors for FBXO32 carrying a Flag epitope (Flag-FBXO32), Myc-CUL1, and GFP-SKP1 were purchased from Origene. Antibodies against FBXO32, cMyc, CUL1, SKP1, HA (Hemagglutinin), GAPDH, and ROC-1 were purchased from Santa Cruz. BECLIN-1, CHMP2B, LAMP2, FBXO32 (mAB), LC3a/B, and p62 were purchased from Abcam, calcineurin A and IkB-α were from Cell Signaling, and IkB-α was from Abcam.