Pinsr

Specific antibodies for pAkt(Ser473)(#4060, 1:5000), pAkt(Thr308)(#4056, 1:2500), Akt(#4691, 1:5000), pINSR(#3024, 1:5000), INSRβ(#3025, 1:5000), p-p70-S6K(#97596, 1:2000), S6K(#9202, 1:5000), pFoxO1(S256)(#9461, 1:2000), FoxO1(#2880, 1:5000), pGSK3β(#5558, 1:5000), GSK3α/β(#5676, 1:5000), p-ERK1/2(T202/Y204)(#9101, 1:5000), ERK1/2(#4695, 1:5000), and Hsp70(#4872, 1:2000) were obtained from (Cell Signaling Technology Inc., Danvers, MA, USA). pIRS1(#09432, 1:2500) and IRS1(#06248, 1:2500) were acquired from (Merck KGaA, Darmstadt, Germany). β-Actin(#A228, 1:5000) was procured from (Sigma-Aldrich). Peroxidase-conjugated secondary antibodies Anti-Rabbit IgG (#NA934, 1:5000) and Anti-Mouse IgG (#NA931, 1:5000) were purchased from (GE Healthcare BioSciences AB, Chicago, IL, USA). Anti-Rabbit IgG Secondary Antibody (Alexa Fluor™ 568, 1:5000) was obtained from (Thermo Fisher Scientific, Waltham, MA, USA).

Protein was extracted from the frozen tissues of animals, including islet, liver, skeletal muscle and white adipose tissue. Protein concentrations were determined by BCA method. Equal amounts of protein (20 μg per lane) were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore). After blocking, the filters were incubated with the primary antibodies. Antibodies including, Akt, p-Akt, InsR (insulin receptor), p-InsR, Acc, p-Acc, HK2 (hexokinase 2), PFKL (6-phosphofructokinase, liver type), PKM1/2 (pyruvate kinase muscle isozyme), PDH (pyruvate dehydrogenase), PDHK1 (pyruvate dehydrogenase kinase isozyme 1) were purchased from Cell Signaling Technology. Antibodies, including PKLR (pyruvate kinase, liver and RBC), PEPCK (phosphoenolpyruvate carboxykinase), CPT1, PTBP1 were purchased from Abcam. Antibodies including GCK (glucokinase), PFK1 (phosphofructokinase 1), G6Pase (glucose-6-Phosphatase), PPARα, PPARδ, PPARγ were purchased from Santa cruz biotechnology. After washes and incubation with the appropriate horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology), the immune complexes were visualized using a chemiluminescence reagent. Western blot results were densitometrically quantified with Quantity One software (Bio-Rad), and the intensity values were normalized to β-actin.