Wy 14643

The following commercial antibodies were used at the indicated dilutions: phospho-GSK-3α (Ser21) (36E9) (1:1,000), total GSK-3α (D80E6, for WB) (1:2,000), total GSK-3α (D80D1, for IF) (1:100), total GSK-3α/β (D75D3) (1:4,000), phospho-GSK-3α/β (1:2,000), histone H3 (1:5,000), GAPDH (14C10) (1:5,000), GFP (D5.1, for IF) (1:500), and secondary antibodies (anti-mouse or rabbit IgG) conjugated with horseradish peroxidase (1:4,000) (Cell Signaling); secondary antibodies (anti-mouse or rabbit IgG) conjugated with Alexa Fluor 488 or 555 (1:100) (Life Technologies); GFP-magnetic beads (Fisher/MBL); total PPARα (1:3,000) (Cayman Chemical); RXRα (D-20) (1:4,000) (Santa Cruz); α-actinin (1:4,000) (sarcomeric) (Sigma-Aldrich). For detection of phosphorylation of PPARα at Ser280, a polyclonal phosphorylation-specific antibody was generated by immunizing rabbits with a phospho-peptide corresponding to residues surrounding Ser280 of PPARα (1:1,000). Antibodies were diluted in either 5% (w/v) BSA or 5% (w/v) non-fat dry milk in 1xTBS/0.5%Tween 20, depending on the level of background intensity. The following reagents were used: WY-14643 and fenofibrate (R & D Systems, Tocris for in vitro experiments); Palmitic acid, Duolink In Situ PLA, and Etomoxir (Sigma-Aldrich).