SUMOylation of EHD3 Modulates Tubulation of the Endocytic Recycling Compartment
Fig 4
A. Lysates of HEK293T cells, transiently transfected with myc–EHD3 or its SUMOylation mutants [EHD3K315R, EHD3K511R, EHD3K(315R+511)R] and HA–SUMO, were coimmunoprecipitated with anti-myc antibody. The immunoprecipitates were subjected to SDS-PAGE and the corresponding blot was probed with anti-HA (to visualize SUMOylation) and anti-myc (to visualize the EHD3 variant) antibodies. In parallel, 5% of the lysates were subjected to SDS-PAGE and the corresponding blot was probed with anti-HA antibody (in order to assess transfection with SUMO1) and anti-myc antibody (to follow presence of transfected EHD3 variant).B. Two micrograms of bacterially purified EHD3 or its SUMOylation mutants (EHD3K315A, EHD3K511A, EHD3K315A/K511A) were incubated with human SUMO1 as detailed in Materials and Methods. The reaction products were subjected to SDS-PAGE and the corresponding blot was incubated with anti-His antibody. IP: immunoprecipitation; WB: western blot.