Rabbit anti phospho foxo1 ser256

Rabbit anti-GLUT4 (no. sc-7938) was obtained from Santa Cruz Biotech (Santa Cruz, CA, USA). Rabbit anti-phospho-AMPK (Thr172) (no. 2531) was purchased from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-PPARα (no. ab8934), and rabbit anti-PPARγ (ab45036) antibodies were purchased from Abcam Inc. (Cambridge, MA, USA). Rabbit anti-FAS (3180), rabbit anti-phospho-Akt (Ser473) (no. 4060), rabbit anti-total AMPK (Thr172) (no. 2532), rabbit anti-phospho FOXO1 (Ser256) (no. 11115), rabbit anti-total-FOXO1 (Ser256) (no. 2880), and rabbit anti-β-actin (no. 4970) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Secondary anti-rabbit antibodies were purchased from Jackson ImmunoRes. Lab., Inc. (West Grove, PA, USA).

The following primary antibodies were used for Western blotting: rabbit-anti-Insulin receptor β (Santa Cruz, #711), rabbit-anti-phospho Akt (Ser473) (Cell Signaling, #4060), rabbit-anti-Akt (Cell Signaling, #9272), rabbit-anti Gsk3β (Cell Signaling, #9315), rabbit-anti-phospho Gsk3β Ser9 (Cell Signaling, #5558), rabbit-anti FoxO1 (Cell signaling, #2880), rabbit-anti-phospho FoxO1 Ser256 (Cell Signaling, #9461), rabbit-anti p70 S6 kinase (Cell Signaling, #2708), mouse-anti-phospho p70 S6 kinase Thr389 (Cell Signaling, #9206), mouse-anti S6 (Cell Signaling, #2317), rabbit-anti-phospho S6 Ser235/236 (Cell signaling, #4856), rabbit-anti 4E-BP1 (Cell Signaling, #9644), rabbit-anti-phospho 4E-BP Thr37/46 (Cell Signaling, #9459), mouse-anti-Gapdh (Abcam #ab8245), rabbit-anti-phospho HSL Ser660 (Cell Signaling, #4126), rabbit-anti-phospho HSL Ser563 (Cell Signaling, #4139), rabbit-anti-HSL (Cell Signaling, #4107), rabbit-anti-PLIN1 (Cell Signaling, #9349) and rabbit-anti-CD36 (Cell Signaling #14347). Secondary antibodies used for western blotting were goat-anti-rabbit IgG-HRP conjugate (Biorad, #1706515) and goat-anti-mouse IgG-HRP conjugate (Biorad, #1706516).

Cells were lysed in RIPA lysis buffer that contained 1 mM PMSF. Total protein concentration was determined using BCA protein assays. After separation on 10% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis gels, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes and then blocked with 5% (wt/vol) non-fat dry milk in Tris-buffered saline that contained Tween 20 for 2 h at room temperature. The PVDF membranes were then incubated with the indicated antibodies, including rabbit anti-β-actin (Bioss) and mouse puromycin antibody 12D10 (Millipore); or rabbit anti-phospho-mTOR (Ser2481) and mTOR, rabbit anti-phosphor-P70S6K (Thr389) and P70S6K1, rabbit anti-phospho-S6 (Ser235/236) and S6, rabbit anti-phospho-4E-BP1 (Thr37/46) and 4E-BP1, rabbit anti-Akt, rabbit anti-phospho-Akt (Ser473), rabbit anti-phospho-Akt (Thr308;), rabbit anti-eIF4E, rabbit anti-phospho-eIF4E (Ser209), rabbit anti-eIF2α, rabbit anti-phospho-eIF2α (Ser251), rabbit anti-FoxO1, and rabbit anti-phospho-FoxO1 (Ser256) (Cell Signaling Technology, Beverly, MA, USA). The primary antibodies were incubated at 4 °C overnight and followed by the incubations of the appropriate secondary antibody (Bioss) for 1 h at room temperature. Protein expression was measured using a FluorChem M Fluorescent Imaging System (ProteinSimple, Santa Clara, CA, USA) and normalized to β-actin expression.