AUF1 p45 is methylated in vitro and in cells. (A) FLAG fusion proteins of the different AUF1 isoforms were expressed in Huh7 cells, immune-precipitated from cell extracts using anti-FLAG-antibody and eluted with FLAG peptide (see Materials and Methods; Friedrich et al. 2014). The presence of PRMT1 in the original extracts (input) and in the eluate was verified by Western blot with an anti-PRMT1-antibody. Vinculin was stained with a Vinculin-specific antibody as an internal control. (B) In vitro methylation assay of AUF1 p45. Thirteen picomoles of recombinant, Escherichia coli generated AUF1 p45 were methylated in vitro by 5 pmol PRMT1 in the presence of 400 pmol of [S-14C] adenosylmethionine as described in Materials and Methods. Samples were taken at the indicated time points and the methylation state analyzed by SDS–PAGE and phosphorimaging. (C) In vitro methylation assay with PRMT1 and different preparations of AUF1 p45. Applied was 5 pmol of AUF1 p45 that was expressed in and purified from E. coli and hence expected to be nonmethylated, and a comparable amount of FLAG–AUF1 p45 that was expressed in and purified from Huh7 cells. Both protein preparations were subjected to an in vitro methylation assay with 5 pmol of purified PRMT1 v1 and 400 pmol [S-14C] adenosylmethionine as a substrate. The samples were taken after 2 h and analyzed by SDS–PAGE and phosphorimaging. (Lower panel) Western blot with anti-AUF1 antibody demonstrating the use of equal amounts of protein in the in vitro methylation assay. One representative experiment is shown. (D) Same experiment as in C except that FLAG–AUF1 p45 was expressed in and purified from the dendritic cell line DC2.4. (E) Same experiment as in C except that FLAG–AUF1 p45 was expressed in and purified from SHSY5Y cells.
