Figure 4.

Generation of the 75-kd SirT1 band by a cathepsin-mediated cleavage event. A, Immunoblots from protein extracts of chondrocytes left untreated (Unt) or treated with tumor necrosis factor α (TNFα), EST and TNFα, or ALLN and TNFα. B, Immunoblots from protein extracts of chondrocytes left untreated, treated with TNFα, or treated with TNFα and ALLN. C, Transient transfections of chondrocytes with either pcDNA control or FLAG-SirT1. Chondrocytes were left untreated, treated with TNFα, or treated with TNFα and ALLN. Immunoblots from protein extracts of the transfected and treated chondrocytes are shown. D, SirT1 activity in chondrocytes left untreated, treated with TNFα, or treated with TNFα and ALLN after immunoprecipitation with a SirT1 N-terminal antibody. SirT1 was immunoprecipitated from chondrocyte extracts, and the immunoprecipitates were processed for SirT1 activity (top) or for immunoblotting (bottom). Immunoblots in A–D are representative of 6 repetitions. E, Relative RNA levels in chondrocytes left untreated (solid bars), treated with TNFα (open bars), or treated with TNFα and ALLN (shaded bars), determined by quantitative polymerase chain reaction with the indicated human primers. Bars show the mean ± SD. * = P < 0.05 versus untreated controls. See Figure 2 for other definitions.