Human Maf1 is phosphorylated and becomes dephosphorylated after rapamycin and MMS treatment.

<p>A) Nuclear extract from a HEK 293 cell line expressing HA-tagged Maf1 either untreated (upper panel), or treated with rapamycin (middle panel) or MMS (lower panel) was incubated with nothing, CIAP with or without phosphatase inhibitors, or phosphatase inhibitors alone, as indicated above the lanes. Tagged Maf1 was visualized with an antibody directed against the HA tag. Lane 5 shows bacterially expressed Maf1, which was loaded on a non-adjacent lane of the same gel. B) Nuclear extract from IMR-90Tert cells transiently expressing HA-tagged Maf1 was prepared in the presence of phosphatase inhibitors prior to immunoprecipitation with affinity-purified anti-RPC1 antibody. Tagged Maf1 was visualized with an antibody directed against the tag (α-HA antibody). Only the fastest migrating form of Maf1 bound to pol III. Arrows: phosphorylated Maf1. The negative control was a mock immunoprecipitation performed with protein A sepharose beads without antibody. Lane 1 shows 1/20 of the input material.