Figure 1
p27 is a substrate of caspases in vitro and in vivo. (a) In vitro cleavage of recombinant wild-type (wt) p27 and a p27-S140E mutant with caspase-3. Samples were taken from the reaction at the indicated time points and hexahistidine (His)-tagged p27 protein was detected by western blotting using anti-p27 antibodies. These antibodies detect the N-terminal fragment of the processing product. − represents recombinant p27 incubated in the absence of caspase-3 for 90 min. Representative blots of three independent biological experiments. (b) Apoptosis was induced in WM35 melanoma cells by irradiation with UV-light (80 J/m2). Cells were collected at the indicated time points and extracts were separated by SDS–PAGE and analyzed for endogenous p27, Skp2, cyclin D1, cyclin E, cyclin A, cyclin B1, cleaved caspase-3 (C3) and β-tubulin. − indicates untreated cells. Representative blots of four independent biological experiments. (c) Apotosis was induced in CEM C7H2 cells by treatment with the glucocorticoid analogue dexamethasone (Dex) at a final concentration of 10−7 M. Cells were collected at the time points indicated and extracts were blotted for endogenous p27, Skp2, cleaved caspase-3, PARP1 and β-tubulin. p27 cleaved by caspase-3 in vitro and extract derived from UV-treated WM35 cells were loaded for size comparisons. Representative blots of three independent experiments are shown. (Co) represents a solvent (ethanol) control; − represents untreated cells.
