Fig. 1.
Serine 75 is a functionally significant site in Maf1 that is phosphorylated in a rapamycin-sensitive manner. (A) HeLa cells transfected with vector or HA-tagged Maf1 were labeled for 3 h with [32P] orthophosphate in the presence of vehicle or rapamycin, as indicated. Panels show autoradiography (Upper) and Western blot analysis (Lower) after immunoprecipitation with anti-HA antibody. (B) Cells were transfected with empty vector or with vector encoding WT, S75A, or S75D Maf1, as indicated, and treated with vehicle or rapamycin. Western blots are shown with antibodies against HA, S75-phosphorylated Maf1, total Maf1, and tubulin. The arrow indicates transfected HA-tagged Maf1; the asterisk indicates endogenous Maf1. (C) Expression of pre-tRNATyr was assessed by real-time qRT-PCR following transfection with empty vector or with vector encoding WT, S75A, or S75D Maf1. Data presented represent levels of pre-tRNATyr after normalization to TFIIB mRNA, with empty vector assigned a value of 1.0. n = 3.