Figure 6.

In vivo methylation of Sam68 is abrogated in prmt1 −/− ES cells. (A) Wild-type ES cells (+/+) as well as ES cells heterozygotes (+/−) or homozygotes (−/−) for a null mutation in the prmt1 gene were harvested in lysis buffer and subjected to immunoblotting by using our anti-PRMT1 and ASYM24 antibodies. Molecular weight markers (in kilodaltons) are shown on the left of each panel. (B) In vivo methylation labeling was performed by metabolically labeling cells with l-[methyl-³H]methionine for 3 h in the presence of cycloheximide and chloramphenicol. The cell lysates were immunoprecipitated with anti-Sam68 (AD1), normal rabbit serum (CTRL), or anti-SMN antibodies. The ³H-labeled proteins in TCLs (10% input) and in immunoprecipitates were separated by SDS-PAGE and visualized by fluorography (lane 1–12; exp. 24 h). Immunoblotting was performed on total cell lysates by using anti-Sam68 (lanes 13–15) and anti-SMN (lanes 16–18) antibodies to confirm equal levels of proteins.