Image studio lite software 5

Cells were lysed in radio-immunoprecipitation assay (RIPA) buffer containing an anti-protease and anti-phosphate cocktail (Thermo Fisher Scientific, Waltham, MA, USA) and electrophoresed on 4–12% polyacrylamide gels under reducing conditions followed by electrotransfer to nitrocellulose membranes. The membranes were incubated with primary antibodies, then washed with phosphate buffered saline (PBS) containing 0.1% Tween-20 and then incubated with species specific IRDye^® 680RD/800CW-conjugated second antibodies. Immunoreactive bands were visualized with the Odyssey Imaging System (LI-COR). Densitometric analysis was performed using the software Image Studio™ Lite Software 5.2 (LI-COR).

Mice were injected with insulin (0.75 U/kg of human insulin) or saline via the tail vein and sacrificed 10 min post-injection. Tissues [liver, epididymal AT (EAT) and muscle] were collected, and analysed by western blot with antibodies to Akt (Cell Signaling Technology #9272), phospho-Akt (Cell Signaling Technology #9271, which detects endogenous levels of Akt only when phosphorylated at Ser 373), and β-actin (Cell Signaling Technology). UCP-1 protein expression in brown fat was determined by western blot analysis with antibodies to UCP-1 (Abcam, Waltham, MA). Tissues were lysed in RIPA buffer (Sigma) containing protease and phosphatase inhibitors (Thermo Fisher Scientific). Lysates were then electrophoresed and transferred to nitrocellulose membranes. The membranes were incubated with primary antibodies, washed with PBS-containing 0.1% Tween-20 and incubated with species specific IRDye®680RD/800CW-conjugated secondary antibodies. The Odyssey Imaging System (LI-COR) was used to visualize the immunoreactive bands, and densitometric quantification performed using Image Studio™ Lite Software 5.2 (LI-COR).

Cells were lysed in RIPA buffer containing anti-protease and anti-phosphatase cocktail (Thermo Fisher Scientific). Cell lysates were electrophoresed on denaturing 4–15% polyacrylamide-SDS gels under reducing conditions and electrotransferred to nitrocellulose membranes. Membranes were incubated with primary antibodies, washed with PBS-Tween-20 0.1% and incubated with species specific IRDye®680RD/800CW-conjugated secondary antibodies. Immunoreactive bands were visualized using the Odyssey Imaging System (LI-COR), according to the manufacturer's instructions (Biosciences). For densitometric analysis, membranes were scanned and quantified using the software Image Studio™ Lite Software 5.2 (LI-COR).