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

FIGURE 4.

FIGURE 4.

Phosphorylation of Y18, Y216, Y395, and Y511 residues of ELMO1 is important for ELMO1-Nck-1 interaction. A, HEK293T cells were transfected with WT ELMO1, or indicated ELMO1 mutant. 24 h after transfection, cell lysates were harvested and the corresponding cell lysate was incubated with purified GST-Nck-1-SH2. ELMO1 bound to GST-Nck-1-SH2 were detected by immunoblot using anti-Myc antibody. Expression of Myc-tagged WT ELMO1 or ELMO1 mutant proteins was assessed by immunoblot using anti-Myc antibody. C, HEK293T cells were transfected with WT ELMO1 or 4YF mutant ELMO1 together with Flag-tagged Nck-1 and HA-tagged Hck. Cell lysates were immunoprecipitated by anti-Myc antibody followed by anti-Flag or anti-4G10 immunoblot. Replicate samples of lysates before IP were analyzed by immunoblot to verify expression of exogenous ELMO1, Nck-1, and Hck proteins using anti-Myc, anti-Flag antibodies, and anti-HA, respectively. B and D, graphic representation of the ratio of Nck-1-bound ELMO1 to total ELMO1. Data are expressed as mean ± S.D. of three independent experiments. *, p < 0.05 versus WT ELMO1 transfected cells.