CD40L triggers posttranslation modification of SMRT and its translocation to the cytoplasm. (A) Western blots were performed using SMRT antibody in Ramos cells exposed to CD40L during the indicated time points. Actin Western blots were performed as a loading control. As previously reported, SMRT is represented by 2 bands, a faster migrating product (SMRT) and a slower band (SMRT-X) indicative of a posttranslational modification. (B) The densitometry of 3 independent SMRT Western blot replicates. The signal of each SMRT Western blot was first normalized by actin levels and then expressed as fold change relative to untreated cells. ■ represents the faster migrating band; , the slower migrating posttranslationally modified form of SMRT. Error bars represent SEM. (C) Ramos cells were transfected with a GFP-SMRT-expressing plasmid and then exposed to CD40L for 2 hours. Immunofluorescence and phase contrast microscopy were performed to determine the cellular localization of transfected SMRT. Rows 3 to 5 show experiments in which transfected Ramos cells were pretreated with the MEK kinase inhibitors PD98059 and UO126 or vehicle, followed by exposure to CD40L.