FIGURE 1.

ELMO1 interacts with Nck-1. A, Myc-tagged ELMO1 and Flag-tagged Nck-1 were co-transfected into HEK293T cells. HEK293T cells lysates were immunoprecipitated with anti-Myc antibody or IgG followed by anti-Flag immunoblot. Replicate samples of lysates before IP were analyzed by immunoblot to verify expression of exogenous ELMO1 and Nck-1 proteins using anti-Myc and anti-Flag antibodies, respectively. B, endogenous Nck-1 co-precipitates with endogenous ELMO1. Lysates of HEK293T cells were immunoprecipitated using anti-ELMO1 antibody or IgG followed by anti-Nck-1 immunoblot. Replicate samples of lysates were analyzed by immunoblot to verify equivalent loading. C, schematic representation of Myc-tagged ELMO1 mutants used. D, HEK293T cells were transiently transfected with ELMO1^WT, or indicated ELMO1 mutant. The corresponding cell lysate was incubated with purified GST-Nck-1-SH2. Bound protein was detected by immunoblot using anti-Myc antibody. Replicate samples of cell lysates were analyzed by immunoblot to verify expression of ELMO1 mutant using anti-Myc antibody. E, Nck-1 interacts directly with ELMO1. Nck-1 and ELMO1 were bacterially produced and purified. His-tagged ELMO1 was incubated with GST or GST-tagged Nck-1-SH2 proteins, and the co-precipitation of ELMO1 was assessed by immunoblotting using anti-ELMO1 antibody. Purified GST-Nck-SH2 or GST added to the reaction was separated by SDS-PAGE and stained with Coomassie Blue (bottom gel).