A1340

The gastrocnemius muscles were segregated and placed in liquid nitrogen for rapid freezing. Frozen samples were pulverized and lysed with 500 µl of the RIPA buffer (Beyotime, China). The lysates were ultrasonicated for 6*3 sec on ice and placed on ice for 45 min. Then the lysates were centrifuged at 4,500 g for 15 min at 4°C and the precipitate was discarded. Protein concentrations in the supernatants were determined using the Bradford reagent (Bio-Rad, Hercules, CA). The protein samples (10 µg) were resolved by 10%-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membrane (Millipore, Billerica, MA, USA). Finally, immunodetection was performed using an enhanced chemiluminescence detection kit (DW101, TransGen Biotech, Beijing, China), then images were captured by Syngene Bio Imaging (Synoptics, Cambridge, UK). The following primary antibodies were used: HSP75 (10325-1-AP, Proteintech, USA), HSP60 (15282-1-AP, Proteintech, USA), HSP10 (sc-376313, Santa Cruz, USA), LONP1 (15440-1-AP, Proteintech, USA), CHOP (15204-1-AP, Proteintech, USA), ATF4 (sc-200, Santa Cruz, USA), Nrf2 (sc-13032, Santa Cruz, USA), Keap1 (10503-2-AP, Proteintech, USA), Foxo3 (10849-1-AP, Proteintech, USA), SOD1 (10269-1-AP, Proteintech, USA), SOD2 (A1340, Abclonal, USA), UCP2 (11081-1-AP, Proteintech, USA), GAPDH (AC002, Abclonal, USA).