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Figure 4


Fig. 4. Ubiquitylation of Dock180 on the plasma membrane. (A) Ubiquitylation of Dock180 in the membrane fraction. Cell lysates of HEK293T cells expressing the indicated plasmids were separated into cytosolic and membrane fractions. The samples were subjected to an in vivo ubiquitylation assay. The expression levels of transfected proteins were analysed using cell lysates of the cytosol and membrane fractions. Anti-E-cadherin antibody was used as a marker for the membrane fraction. CL, cell lysate. (B) Localisation of Dock180 and ubiquitin in cells. HEK293T cells (upper panels) and Cos-7 cells (lower panels) were transiently transfected with Flag-Dock180 or Dock180-RFP and HA-ubiquitin expression plasmids. After incubation for 36 hours, cells were stained with anti-HA tag antibody and analysed by confocal microscopy. (C) Ubiquitylation of Dock180 in the membrane fraction under EGF stimulation. HEK293T cells expressing the indicated plasmids were stimulated by EGF or not. Cell lysates were subjected to in vivo ubiquitylation assay.





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