Figure 7. SCML2B stabilizes p21.

(A) Western blot analysis of p21, p27, and actin, using whole cell extracts from U2OS cells treated with two different siRNAs (#1, #2) for SCML2 or a control siRNA (C) for 72 h, and then incubated in the presence of 5 µM MG132 or DMSO. (B) Quantification of the densitometric analysis for p21 and p27, from three different experiments performed as in (A). The intensity was corrected for the expression of actin, and the ratio of the levels after MG132 treatment relative to those of DMSO treatment is shown for each siRNA. *p<0.01 (C) U2OS cells were transfected with a control siRNA and plasmid (Control), with an siRNA against SCML2 and a control plasmid (si#2) or an siRNA against SCML2 and a plasmid expressing SCML2B resistant to the siRNA (si#2+SCML2B) for 48 h. Cells growing asynchronously (top panel) or released from an arrest in mitosis (5 h, middle panel, and 8 h, bottom panel) were then incubated with 25 µg/ml cycloheximide, and whole cells extracts were analyzed at different time points by Western blot with antibodies specific for p21. Densitometric quantification of the levels of p21 from two different experiments, each of them loaded in duplicate, is shown. The data were fitted to a one phase decay equation.