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. 2014 Jun 13;289(33):23112–23122. doi: 10.1074/jbc.M114.549550

FIGURE 6.

FIGURE 6.

Nck-1 promotes the interaction between ELMO1 and active RhoG. A, schematic representation of WT ELMO1, ARM1, or ARM2 mutant of ELMO1 used. B, Myc-tagged WT ELMO1, ELMO1 ARM1, or ARM2 mutant was transfected into HEK293T cells, respectively. Cell lysates were incubated with purified GST-Nck-1-SH2. Bound protein was detected by immunoblot using anti-Myc antibody. Expression of Myc-WT ELMO1 or ELMO1 mutant proteins was assessed by immunoblot using anti-Myc antibody. C, Myc-tagged ELMO1 and HA-tagged RhoGV12A were co-transfected with Flag-tagged WT Nck-1, R308K, or SH3–1−,2−,3− into HEK293T cells. Cell lysates were immunoprecipitated by anti-Myc antibody followed by anti-HA, anti-Flag immunoblot. Expression of ELMO1, Nck-1, or RhoGV12A proteins was assessed by immunoblot using anti-Myc, anti-Flag, or anti-HA antibody. D, Myc-tagged WT ELMO1 or Y4F mutant was co-transfected into HEK293T cells with HA-tagged RhoGV12A. Cell lysates were immunoprecipitated by anti-HA or anti-IgG antibody followed by anti-Myc immunoblot. Expression of exogenous ELMO1 or RhoGV12A proteins was assessed by immunoblot using anti-Myc or anti-HA antibody.