Figure 3.

The 75-kd SirT1 fragment is a posttranslationally modified form of SirT1. A, Outline of the FLAG-tagged SirT1 expression plasmid and the regions detected by the N-terminal–specific and C-terminal–specific antibodies. B, Transient transfections of human chondrocytes with the FLAG-tagged SirT1 plasmid or a pcDNA control plasmid. The cells were left untreated (Unt) or were treated with tumor necrosis factor α (TNFα), and protein extracts were analyzed by immunoblotting with the indicated antibodies. Immunoblot is representative of 6 repetitions. C, Cleavage site in SirT1. The area between residues 520 and 540 delineates the cleavage site within the carboxy-terminal domain of SirT1, based on the relative size of the 75-kd fragment. See Figure 2 for other definitions.