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Fig. 1. Elmo1 increases the stability of Dock180 protein. (A) Left panels; HEK293T cells were transiently transfected with mammalian expression plasmids for Flag-Dock180 alone or Flag-Dock180 and myc-Elmo1. After 48 hours, cells were lysed and analysed by immunoblotting (IB) with anti-Flag tag, anti-myc tag and anti-actin antibodies. The active form of Rac was detected in a pull-down assay. Right panels: HEK293T cells were transfected with mammalian expression plasmids for Flag-Crk II alone or Flag-Crk II and myc-Elmo1, and cells were analysed in the same way as for the left panels. (B) For pulse-chase analysis for Dock180, HEK293T cells transiently expressing Dock180 alone (-) or Dock180 and Elmo1 (+) were labelled for 1 hour and then chased for the time indicated at the top of the panel. (C) The signal intensity of Dock180 was measured and shown as a bar graph with the average and standard errors of three independent experiments; **P<0.05. (D) mRNA levels of exogenous Dock180. RT-PCR analysis was performed using mRNA extracted from HEK293T cells transiently expressing Dock180 alone or Dock180 and Elmo1. GAPDH is a control for the amounts of PCR templates. T/F, transfection; RT, reverse transcriptase. (E) HT1080 cells were transfected with siRNAs for negative control, scramble control and Elmo1#3. After incubation for 96 hours, cell lysates were subjected to immunoblotting for detection of Dock180, Elmo1 and actin. (F) The signal intensity of Dock180, normalised by the intensity of actin, is shown as a bar graph with the average and standard errors of three independent experiments; **P<0.05. (G) mRNA levels of endogenous Dock180. RT-PCR analysis was performed using the same mRNA extracts as E. GAPDH is a control for the amount of PCR template. (H) HEK293T cells were transfected with negative control and Elmo1#2 and cells were analysed in the same manner as E.
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