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Fig. 3. Elmo1 suppresses ubiquitylation of Dock180. (A) A schematic representation of wild-type and mutant forms of Elmo1 used in the study. DUF609, domain of unknown function; FL, full length; LZ, leucine zipper; PH, pleckstrin homology; PxxP, polyproline-rich motif. (B) Association between Dock180 and Elmo1 in HEK293T cells. Cells were transiently transfected with the indicated plasmids, and lysates were immunoprecipitated with anti-myc tag antibody, and then probed with anti-myc tag and anti-Flag tag antibodies. TCL, total cell lysate. (C) Ubiquitylation of Dock180 in HEK293T cells in the presence of Elmo1 and its mutants. 24 hours after transfection with the indicated plasmids, cells were lysed and subjected to in vivo ubiquitylation assay. (D) The urea-reversal immunoprecipitation was performed using the same lysates as C. The IP ratio represents the relative signal intensity of immunoprecipitated Dock180 in lane 1 as 1.0 (bottom of the panel). (E) The ubiquitylation of Dock180 in the absence or presence of Elmo1 and GST. The immunoprecipitation was performed using anti-Flag tag antibody. (F) The ubiquitylation of Dock180 and its mutant. Lysates from HEK293T cells expressing the indicated plasmids were immunoprecipitated with anti-Dock180 antibody at in vivo ubiquitylation assay. The IP ratio represents the relative signal intensity of immunoprecipitated Dock180 in lane 1 as 1.0.
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