HeLa cells were transfected for 24 h with MARCH8 GFP construct and treated with 400 μM bafilomycin A1 for 4 h. The cells were incubated at 18°C with anti-TfR antibody (5E9C11) for 1 h. Alternatively, a parallel dish was incubated at 37°C with 15 min with HBSS, loaded with anti-TfR antibody (5E9C11) for 30 min, and chased with 15 min with HBSS. Control cells were incubated at 37°C with control mouse immunoglobulin G (IgG). Immunoprecipitates obtained were processed for immunoblot for ubiquitin. Cartoon in (B) shows the different steps for the TfR antibody internalization. Data are mean ± SD (n= 3; graphs represent means from three independent experiments). Two-tailed paired t test,**p< 0.01; ns, not significant.
