In vitro VEGFR-2 Methylation Assay

In vitro methylation assay was performed as described with minor modification (Hartsough et al., 2013 (link)). Briefly, VEGFR-2 was immunoprecipitated from HEK-293 cells expressing VEGFR-2 alone or co-expressing VEGFR-2 and PRMT4 were incubated with the methylation reaction buffer ((50 mM Tris pH 8.5, 20 mM KCl, 10 mM MgCl2, 1 mM β-mercaptoethanol, and 100 mM sucrose) plus 1 μL of adenosyl-L-methionine, S-[methyl-3H] (1 mCi/mL stock solution, Perkin Elmer) at 30°C. In some experiments, a purified GST-PRMT4 also added into the reaction mix. The reaction stopped by adding 2× SDS loading buffer after 1 h and was resolved on SDS-PAGE. The separated protein samples were then transferred from the gel to a polyvinylidene difluoride (PVDF) membrane, sprayed with EN3HANCE (Perkin Elmer) and exposed to X-ray film.