Dylight 800 labeled secondary antibody

Total cell protein lysates were extracted with RIPA (Radio Immunoprecipitation Assay) lysis buffer which contained protease inhibitor (HX1863, Beijing Huaxing Bochuang Gene Technology Co., Ltd, Beijing, China) and protein phosphatase inhibitor cocktail (HX1864, Beijing Huaxing Bochuang Gene Technology Co., Ltd). Approximately 60 to 100 μg of total cell lysates were loaded per well, and GAPDH was run as a loading control. Protein samples were resolved on 10% SDS-PAGE gels and transferred to PVDF membranes (Millipore, USA). Membranes were blocked in tris-buffered saline containing 5% (wt/vol) nonfat dry fat at room temperature for 1 h and then incubated against corresponding primary antibodies at 4 °C overnight. After washing 3 times with TBST solution for 10 min, the membranes were incubated with DyLight 800-labeled secondary antibody (#5151, Cell Signaling Technology, Beverly, MA, USA) at room temperature for 1 h. The signal was detected using Odyssey Clx (LiCor Biosciences, Lincoln, NE, USA). Band density was quantified by ImageJ software.

Frozen liver and muscle tissues obtained from Experiment 1 and 2 were rapidly powdered in liquid nitrogen and lysed in RIPA buffer with protease‐ and phosphatase‐inhibitors, followed by sonication and centrifugation. A sum of 60 μg protein from each sample was loaded, separated by 10% SDS polyacrylamide gels, transferred to a polyvinylidene fluoride membrane (Millipore). The blotted membranes were incubated with corresponding primary antibodies overnight at 4 °C (Table S2, Supporting Information). After three washes, the membranes were incubated with DyLight 800‐labeled secondary antibody (Cell Signaling Technology, 5151) for 1 h at room temperature. Band densities were detected with the Odyssey Clx (Gene Company Limited, Hong Kong, China) and quantified using the ImageJ software.