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. 2010 Jul 6;30(17):4245–4253. doi: 10.1128/MCB.00218-10

FIG. 3.

FIG. 3.

Characterization of the in vivo methylation activity of N6amt1 in transfected HEK-293T cells. (A) Detection of transfected proteins by Western analysis. HEK-293T cells were transiently transfected with the eRF1-FLAG expression construct alone (“Ctrl” sample) and with the constructs for HA-N6amt1, Trm112-myc, and FLAG-eRF3 (“Plus” sample). Epitope-specific antibodies were used in the detection. (B) Increased methylation of eRF1 when cotransfected with HA-N6amt1, Trm112-myc, and FLAG-eRF3. The ectopic eRF1 was immunoprecipitated from the cell extract using anti-FLAG M2 beads prior to Western analysis. Bacterial recombinant eRF1 protein samples, unmethylated and methylated in vitro, were loaded as a control to monitor preferential detection of the methylated form by the anti-methyl-eRF1 (α-methyl-eRF1) antibody (lanes 1 and 2). Different amounts (40, 50, and 60 ng) of the “Plus” eRF1 sample were loaded for quantification of the methylated eRF1. The antibodies used are indicated to the right. α-eRF1, anti-eRF1 antibody. (C) Western detection of N6amt1 and its cofactors transfected into HEK-293T cells for the methylation analysis on endogenous eRF1. Epitope-specific antibodies were used. (D) MALDI-TOF MS analysis for Gln185 methylation of endogenous eRF1 from HEK-293T cells transfected with N6amt1 and its cofactors (“Plus” sample). The endogenous eRF1 protein was immunopurified with anti-eRF1 polyclonal antibodies from transfected HEK-293T cells, separated, and in-gel digested with chymotrypsin prior to MALDI-TOF MS analysis. The peak at m/z 1,677.69 corresponds to methylated peptides containing Gln185 (Fig. 1C). The peak for unmethylated peptide was not detected. (E) N6AMT1 depletion in knockdown cell line N6-A9 verified by real-time PCR. siRNA expression was induced by addition of doxycycline. H1-B4 is a control cell line harboring an empty siRNA vector. (F) N6AMT1 depletion in knockdown cell line N6-A9 verified by Western blotting. (G) MALDI-TOF MS analysis for Gln185 methylation of FLAG-eRF1 in H1-B4 cells. α-FLAG, anti-FLAG antibody; α-β-actin, anti-β-actin antibody. (H) MALDI-TOF MS analysis for Gln185 methylation of FLAG-eRF1 in N6-A9 cells.