Anti mouse igg from sheep

Western blotting was performed with a chemiluminescence detection system. Protein from normal human chondrocytes was lysed using 6 M urea/2% SDS buffer. Cell lysates were sonicated at 4°C and protein concentrations were determined using a Pierce BCA Protein Assay kit (Pierce Biotechnology, Rockford, IL, USA). The proteins were separated on 4–20 % SDS-polyacrylamide gels and transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA), blocked with 5% dry milk or 5% BSA in Tris buffered saline–Tween (TBST), and blotted with rabbit polyclonal antibody specific for LC3 (1:1000; MBL International, Woburn, MA; Cat. Number PM036), or p-Akt (Ser473) or p-rbS6 (1:2000; Cell Signaling Technology, Beverly, MA; Cat. Numbers #4060 and #4858, respectively) or mouse antibody Tubulin (1:2000; Sigma, St. Louis, MO, USA; Cat. Number T9026) for 1 hour. The membranes were then incubated with horseradish peroxidase (HRP)–conjugated anti rabbit IgG (from donkey) or anti mouse IgG (from sheep) (1:5000; GE Healthcare, UK; Cat. Numbers NA934 and NA931, respectively) for 1 hour. Afterward, the membranes were washed 3 times with TBST and developed using enhanced chemiluminescence substrate (Pierce Biotechnology).