Research Paper Volume 6, Issue 9 pp 755—769

Figure 1. Mechanisms of prelamin A degradation. HEK293 cells were transiently transfected with prelamin A constructs yielding wild-type lamin A (LA-WT), non-farnesylatable unprocessable prelamin A (LA-C661M), or farnesylated (partially processed) prelamin A (LA-L647R). (A) Transfected cells were subjected to Chloroquine diphosphate (chloroquine) to block lysosome-mediated degradation. Proteins separated on 8% SDS-PAGE were subjected to Western blot analysis. LA-C661M was accumulated in chloroquine-treated cells, indicating lysosomal degradation of non-farnesylated prelamin A. The presence of LC3-B2 band indicates activation of the lysosomal degradation pathway. (B) Phosphorylation of Serine 404 (P-Ser404) is detected in LA-WT and LA-C661M, while LA-L647R is minimally phosphorylated. LA-S404A is a non-phosphorylatable lamin A mutant and was tested as a negative control for anti-P-Ser404 antibody, LA-S404D is a phosphomimetic mutant for P-Ser404. Densitometric values of P-Ser404 labeled bands normalized to FLAG-labeled bands are reported in the graph as mean values of triplicate experiments +/− Standard Deviation. Asterisks indicate significantly different values relative to LA-WT (p< 0.05) determined by Student's T test. (C) To test rapamycin activity on prelamin A mutants, cells were treated with rapamycin (rapamycin) for the indicated time periods. Farnesylated prelamin A (LA-L647R) was selectively degraded (upper panel). Phosphorylation of Serine 404 (P-Ser404) is shown in the lower panel. (D) The densitometric values of anti-prelamin A labeled bands detected in chloroquine or rapamycin-treated cellular lysates were measured. Data are reported as percentage of the densitometric value of each corresponding untreated sample (designated as 100%). The mean values of triplicate experiments +/− Standard Deviation are reported. Actin has been labeled as a loading control. Prelamin A was detected by anti-prelamin A (Santa Cruz Sc-6214) antibody or by anti- FLAG antibody (Sigma-M2).