Figure 5
From: The interactome of a family of potential methyltransferases in HeLa cells
Confirmation of METTL9 interactor and enzymatic activity of GFP-METTLs. (a,b) Validation of interaction between METTL9 and CANX by co-IP. (a) CANX is detected in GFP-METTL9 IP (a, lane 5) but not GFP IP (a, lane 2). 10 μl of GFP trap and 2 mg of whole cell extract were used. 20 μg of Input material were loaded for a comparison. (b) GFP-METTL9 (left panel, line 2) but not GFP (right panel, line 3) can be detected by immuno-blot with GFP antibody in the CANX IP. 10 μg of calnexin antibody and 4 mg of whole cell extract were used. 200 μg of Input material were loaded for comparison. No antibody (beads alone) used as control. (c) In vitro methyltransferase assays demonstrating that our GFP-METTL8 and GFP-METTL16 purifications have the expected RNA methyltransferase activity. GFP (as a control) and GFP-fusion proteins were purified from corresponding DOX-induced HeLa FRT cell lines and used in an in vitro methyltransferase assay on total RNA from HeLa cells as a substrate and 3H-SAM as a methyl-donor. After purification of the RNA, counts per minute (CPM) were quantified by liquid scintillation counting. Ratio of CPM measured for reactions with GFP-METTL fusion proteins relative to GFP control are plotted. Data are shown as mean ± SD from three replicates.
