Midi gel system

Dilutions of whole-tissue extracts with NuPAGE® 4 × LDS Sample Buffer were loaded on 10% NuPAGE® Bis-Tris gels (Life Technologies). Proteins were separated by gel electrophoreses running on an Invitrogen Midi Gel system and transferred to polyvinylidene difluoride (PVDF) membranes on an Invitrogen semi-dry apparatus at 20 V for 1 h. The membranes were blocked with 5% non-fat dry milk in TBS (10 mM Tris, 150 mM NaCl, pH 7.4) for 1 h at room temperature. Primary antibodies (1:500 dilution for CD36, 1:1000 dilution for Erk1/2, pErk1/2, total Akt, and 1:2000 dilution for pAKT^S473) were incubated overnight at 4 °C in TBST (TBS with 0.1% Tween 20) with 5% BSA. After four 5 min washes in TBST, the secondary antibody (Odyssey IRDye 800CW goat anti-rabbit-HRP, Li-Cor Biosciences) was incubated in a 1:10000–1:15000 dilution in TBST with 5% BSA for 1 h at room temperature followed by four 5 min washes in TBST. Detection of immuno-reactive bands was performed by image scan using a LI-COR Odyssey Imaging System (LI-COR Biosciences, Lincoln, NE).

Dilutions of whole-tissue extracts with NuPAGE 4 × LDS sample buffer were loaded on 10% NuPAGE Bis-Tris gels (Life Technologies, Camarillo, CA, USA). Proteins were separated by gel electrophoreses running on an Invitrogen Midi Gel system and transferred to polyvinylidene difluoride (PVDF) membranes on an Invitrogen semi-dry apparatus at 20 V for 1 h. The membranes were blocked with 5% nonfat dry milk in TBS (10 mm Tris, 150 mm NaCl, pH 7.4) for 1 h at room temperature. Primary antibodies (1:500 dilution for CD36, 1:1000 dilution for Erk1/2, pErk1/2, total Akt, and 1:2000 dilution for pAKT ^S473) were incubated overnight at 4° C in TBST (TBS with 0.1% Tween 20) with 5% BSA. After four 5 min washes in TBST, the secondary antibody (Odyssey IRDye 800CW goat anti-rabbit-HRP, Li-Cor Biosciences, Lincoln, NE, USA) was incubated in a 1:10000-1:15000 dilution in TBST with 5% BSA for 1 h at room temperature followed by four 5 min washes in TBST. Detection of immuno-reactive bands was performed by image scan using a LI-COR Odyssey Imaging System (LI-COR Biosciences).