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. 2021 Dec 18;22(24):13593. doi: 10.3390/ijms222413593

Figure 1.

Figure 1

TRMT112 interacts with seven MTases in mammalian cells. (A) Generation of stable U2OS cell lines expressing the TRMT112-E2Tag protein. Protein expression was detected by Western blot analysis using antibodies against TRMT112 and E2Tag epitope tag. Images of results with anti-TRMT112 depict both recombinant (upper band) and endogenous protein (lower band marked with asterisk *). (B) Immunoprecipitation analysis of TRMT112 from stable cell lines using E2Tag antibodies. (C) Experimental scheme of the SILAC assay coupled co-immunoprecipitation assay. (D) MTases interacting with TRMT112 pulled down in SILAC screen. H/L ratio shows the relative enrichment of proteins pulled down with TRMT112 compared to mock control. (E) The endogenous TRMT112 protein interacts with all seven MTases identified with SILAC assay. EGFP-fused MTases were expressed in U2OS cells and immunoprecipitated using GFP-Trap beads followed by Western blot analysis with EGFP and TRMT112 antibodies. (F) TRMT112-EGFP fusion protein was expressed together with epitope tagged-MTases in U2OS cells, and GFP-Trap beads were used to immunoprecipitate TRMT112-EGFP protein. Western blot analysis was performed with antibodies against E2Tag to detect MTases, and EGFP for analysing TRMT112-EGFP protein levels in immunoprecipitated probes.