Anti akt1 2 3 h 136

Cell lysates were prepared with the ProteoJET Mammalian Cell Lysis Reagent (Fermentas, Burlington, Canada). Twenty µg of the cell lysate isolated from Rin-5F or islet cells or 20 µg proteins precipitated from the cell culture medium after cell treatment were separated by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto a polyvinylidene difluoride (PVDF) membrane. Immunoblot analysis was performed using anti-PARP-1 (H-250; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-PAR (H10; Alexis Biochemicals, San Diego, CA, USA), anti-Akt 1/2/3 (H-136; Santa Cruz Biotechnology), anti-pAkt 1/2/3 (Ser-473-R; Santa Cruz Biotechnology), anti-ERK 1/2 (K-23; Santa Cruz Biotechnology), anti-pERK (E-4; Santa Cruz Biotechnology) antibodies. Blots were probed with horseradish peroxidase-conjugated secondary antibodies. Staining was performed by the chemiluminescent technique according to the manufacturer's instructions (Amersham Pharmacia Biotech, Amersham, UK). If different set of samples were run on different gels, immunoblot analysis was performed in the same time, under the same conditions. All membranes were exposed under the same film and thus exposure and development did not influence the obtained results.

Proteins or cell lysates were electrophoresed by SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with 5% skimmed milk in TBST (50 mM Tris-HCl (pH 7.4), 150 mM NaCl and 0.1% Tween20) for 1 h, then incubated with primary antibodies and secondary antibodies and detected using ECL system (Thermo). The dilutions of the primary antibodies were anti-RXRα (ΔN197, Santa Cruz) in 1:1,000, anti-PARP (H-250, Santa Cruz) in 1:3,000, anti-p85α (Millipore) in 1:1,000, anti-p-AKT (D9E, Cell Signaling Technology) in 1:1000, anti-AKT1/2/3 (H-136, Santa Cruz) in 1:1,000, anti-β-actin (Sigma) in 1:5,000, anti-c-myc (9E10, Santa Cruz) in 1:3,000, anti-HA (F-7, Santa Cruz) in 1:3,000, anti-Flag (F1804, Sigma) in 1:3,000. Images of all uncropped western blots can be found in Supplementary Figs 10–14.