αb crystallin

Rat's skeletal muscles of each group (n = 5; half muscle, 100–200 mg) at 2- and 5-day post-injury were lysed with the appropriate buffer plus a mixture of protease and phosphatase inhibitors (Sigma Aldrich). Homogenates were all centrifuged at 14,000 g for 15 min at 4°C. Equal amounts of muscle proteins (15–20 µg) were separated by SDS-PAGE and transferred onto PVDF membranes (Amersham Pharmacia Biotech). Membranes were incubated overnight with the following primary antibodies: MyoD1 (1: 2,000), SRF (1: 500), myogenin (1: 2,000), phospho-ERK1/2 (1: 1,000), Hsp27 (1: 2,000), Bax (1∶1,000) and Akt (Santa Cruz) (Santa Cruz Biotechnology); phospho-p38MAPK (1: 1,000), p38 (1: 1,000), caspase 3 (1: 1,000), phospho-NF-κB p65 (Ser536) (1: 1,000), Bcl-2 (1: 1,000), phospho-Akt (Ser473) (1: 1,000), phospho-Hsp27 (Ser82) (1: 1,000) and p42 MAP Kinase (1: 1,000) (ERK2) (Cell Signaling); Hsp70/72 (1∶1,000), αB-crystallin (1: 2,000) and S59 phospho-αB-crystallin (1: 2,000) (Enzo Life Science); GAPDH (1: 3,000) (Millipore). Blots were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (1: 15,000) (Millipore), and proteins were visualized by chemiluminescence (EuroClone). Bands were quantified by Image J software. The expression of GAPDH was used as a normalizing control. Phosphorylated isoform was normalized on the amount of its total protein.