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. 2018 Jan 26;293(11):4047–4055. doi: 10.1074/jbc.RA117.001260

Figure 2.

Figure 2.

RNF145 is an ER-localized ubiquitin ligase. A, predicted topology of RNF145. YLYF, the amino acids from 81 to 84 of RNF145. B, subcellular localization of RNF145. HeLa cells were transfected with a plasmid encoding RNF145-FLAG and stained with anti-FLAG and anti-calnexin antibodies. Scale bar = 10 μm. C, in vitro ubiquitination assay showing that RNF145 (511–663) possesses E3 activity. Recombinant proteins, including E1, E2 (Ubc7), FLAG-ubiquitin (FLAG-Ub), RNF145 (511–663), and gp78 (309–643), were added to the reaction system as indicated. After incubation at 37 °C for 15 min, samples were subjected to SDS-PAGE, followed by immunoblot (IB) analysis. D, in vitro ubiquitination assay comparing RNF145 (511–663) and RNF145 (511–663) (C537A). Experiments were carried out as described in C.