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

FIGURE 2.

FIGURE 2.

The SH2 domain of Nck-1 is sufficient to bind to ELMO1. A, schematic representation of Nck-1 mutants used. B, lysates from Myc-tagged WT ELMO1-transfected cells were incubated with purified indicated GST-Nck-1 fusion protein, respectively. Bound protein was detected by immunoblot using anti-Myc antibody. GAPDH was used to verify equivalent loading. GST or GST-bound fusion proteins added to the reaction were separated by SDS-PAGE and stained with Coomassie Blue (bottom gel). C, Myc-tagged ELMO1 was transfected into HEK293T cells with Flag-tagged WT Nck-1 or indicated Nck-1 mutants. Exogenous ELMO1 was immunoprecipitated (IP) using anti-Myc antibody. The presence of Nck-1 in the immunocomplexes was detected by immunoblot using anti-Flag antibody. Replicate samples of lysates before IP were analyzed by immunoblot to verify expression of exogenous ELMO1 and Nck-1 mutant proteins using anti-Myc and anti-Flag antibodies, respectively.