Fig. 6. Reduced phosphorylation of serine-404 in progerin.

A. Western blot comparing the levels of serine-404 phosphorylation in human versions of lamin A, farnesyl-prelamin A (f-PreA), and progerin (Prog) expressed in mouse SMCs. Serine-404 phosphorylation was detected with an antibody against human lamin A/C phosphoserine-404. The specificity of the antibody was evaluated using extracts from SMCs expressing an S404A human lamin A mutant and Lmna^−/− SMCs. The levels of expression of the human nuclear lamin proteins were measured with an anti-human lamin A/C antibody. Actin was measured as a loading control. The arrow points to a nonspecific band produced with the human lamin A/C phosphoserine-404 antibody. B. The bar graph compares the levels of serine-404 phosphorylation in the human versions of lamin A, lamin A-S404A, Prog, and f-PreA. Serine-404 phosphorylation was normalized to the level of human lamin protein expression. Mean ± SEM (n = 3). Student’s t test. *, P < 0.05. C. Western blot comparing the effects of progerin expression on the phosphorylation of serine-404 in human farnesyl-prelamin A. Human farnesyl-prelamin A synthesis was induced by transient transfection of human-prelamin A (hPreA) in Zmpste24^−/− SMCs or Zmpste24^−/− SMCs expressing progerin (ZmpKO hProg). Serine-404 phosphorylation in human farnesyl-prelamin A and progerin was detected as described in panel A. The arrow points to a nonspecific band produced with the human lamin A/C phosphoserine-404 antibody. D. The bar graph compares the effects of progerin expression on the levels of serine-404 phosphorylation in farnesyl-prelamin A. The levels of serine-404 phosphorylation were measured as described in panel B. Mean ± SEM (n = 3). ANOVA. *, P < 0.05. ns, not significant.