Figure 1A: For in vivo-methyltransferase (MT) assay, HEK293 cells were transfected with either empty vector (e.v., control) or EGFP-tagged p14ARF-containing plasmid. Subsequently, cells were treated with the global methyltransferase inhibitor adenosine dialdehyde, AdOx (+) or left untreated (-) for 72 h and then cultured in the presence of L-[3H-methyl]-methionine. Cell lysates were subjected to α-GFP immunoprecipitation (IP), then assayed by fluorography (upper panel) and immunoblotting using α-GFP antibody (lower panel). EGFP-epitope tagged p14ARF typically migrated as a doublet band, indicated by the bracket. Corresponding Appendix Fig. S1 confirms p14ARF overexpression in the cell lysates and hypomethylation caused by AdOx treatment. B: Recombinant GST-tagged substrates (p14ARF, GAR) and PRMT1 / PRMT4 enzyme purified from bacteria or PRMT5 overexpressed/immunoprecipitated from HeLa cells (HA-tagged PRMT5/Myc-tagged MEP50) were subjected to in vitro-methyltransferase (MT) assays in the presence of [14C-methyl]-SAM. Reactions were separated by SDS-PAGE, blotted and assayed by autoradiography. GST-GAR, histone H3 and bulk histones served as a positive control for PRMT1, PRMT4 and PRMT5 activity, respectively. The two depicted negative controls (-) for PRMT1 are identical. The asterisks indicate the methylated p14ARF protein. Corresponding autoradiography results show representative images and derive from the same blots and exposure times with white lines indicating where tracks were cut. Size markers (in kDa) are shown on the right. C: Recombinant GST-tagged p14ARF purified from bacteria and Flag-tagged PRMT1, PRMT4 or PRMT5 enzyme purified from baculoviral infected Sf9 cells were subjected to in vitro-methyltransferase (MT) assays in the presence of [14C-methyl]-SAM. Reactions were separated by SDS-PAGE, blotted and assayed by autoradiography. Histone proteins H4, H3 and bulk histones served as a positive control for PRMT1, PRMT4 and PRMT5 activity, respectively. The asterisk indicates the methylated p14ARF protein. Size markers (in kDa) are shown on the right. D: GST-tagged full length ORF (aa 1-132) and deletion constructs (aa 1-64, aa 65-132 and aa 31-132) of p14ARF as well as GST alone were subjected to in vitro-MT assays in the presence of GST-PRMT1 as described in (B). Methylation activities were detected by autoradiography (upper panel). Asterisks indicate methylated p14ARF proteins, whereas the arrowhead marks p14ARF-unrelated background signals (likely deriving from PRMT1 automethylation). Amounts of p14ARF proteins and GST alone were visualized by immunoblotting using α-GST antibody (lower panel). Asterisks highlight the expected size and location of the different proteins. Size markers (in kDa) are shown on the left. E: NLS/NoLS of p14ARF (aa 85-101) is depicted at the top, with the PRMT1-methylated arginine residues distinguished in green and mutations to glycine (G), phenylalanine (F) or lysine (K) in red. For mass spectrometry, GST-tagged p14ARF (full-length protein) was in vitro methylated by PRMT1, separated by SDS-PAGE, in-gel digested with trypsin and analyzed by LC-MS/MS. Fragment ion spectrum resulted from the doubly charged precursor ion of the R99-methylated (upper panel) and the R96-methylated (lower panel) p14ARF peptides showing y ions and b ions by consecutive fragmentation reactions. Relevant ions were labeled according to the accepted nomenclature. Dimethylation of R99 was confirmed by detection of methylated N-terminal b-ions and unmethylated C-terminal y-ions. Dimethylation of R96 was detected in the mass of the b7, y6 and y7 ions. F: Full-length GST-tagged wild type (wt) and RG mutant p14ARF protein, GST alone as well as GST-PRMT1 were purified from bacteria, as visualized by SDS-PAGE and Coomassie Blue staining (left and middle panel), with asterisks indicating the corresponding PRMT1 and p14ARF protein bands. GST and GST-p14ARF proteins (wt, RG) were subjected to in vitro-MT assays in the presence of GST-PRMT1 as described in (B). Methylation activities were detected subsequent to protein blotting by autoradiography (upper right panel). The asterisk highlights the methylated p14ARF wt protein, whereas the arrowhead indicates p14ARF-unrelated background signals (likely deriving from PRMT1 automethylation). Amounts of p14ARF proteins were detected by immunostaining of the autoradiographed blot using α-p14ARF antibody (lower right panel). Size markers (in kDa) are shown on the left. G: U2OS cells were transfected with the indicated plasmids (e.v., empty vector/control). Immunoprecipitation (IP) from cell lysates was performed using α-Myc antibody (exogenous PRMT1) or IgG as negative control. IP reactions and input lysates were analyzed by immunoblotting using the indicated antibodies.