Sc 13132

Muscle proteins were extracted from homogenizing LT muscles (about 0.12 g) in radio immunoprecipitation assay buffer (1.2 mL) at 45 min and 24 h post-mortem. Proteins were quantified and normalized using Coomassie staining. Muscle proteins (5 μL) were separated through 12% SDS-PAGE gel using a Mini-PROTEAN system (Bio-Rad Laboratories Inc., Richmond, CA, USA) and then transferred to polyvinylidene difluoride membranes (GE Healthcare Ltd., Freiburg, Germany). Transferred membranes were blocked with 5% non-fat dry milk powder in Tris-buffered saline/Tween. For Western blot analysis, the following primary antibodies were used: αβ-crystallin (1:10,000 dilution; ab13496, Abcam Ltd., Cambridge, UK), HSP20 (1:1000 dilution; ab13491, Abcam Ltd.), and HSP27 (1:3000 dilution; sc-13132, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). HSP27 antibody detects 27 kDa (intact form) and 22 kDa (degraded form) proteins. The secondary antibody used was anti-mouse immunoglobulin G (IgG) horseradish-peroxidase-linked antibody (1:3000 dilution; Cell Signaling Technology Inc., Danvers, MA, USA). A WesternBright ECL Kit (Advansta Inc., Menlo Park, CA, USA) was used to detect proteins and images were taken using the ImageQuant LAS 500 (GE Healthcare Ltd., Freiburg, Germany). Each protein band was analyzed using one-dimensional image analysis software (Eastman Kodak Co., Rochester, NY, USA).