Figure 3.
Figure 3. SHIP2 interacts with the EVH1 domains of Mena and VASP via the FPPPP motif. (A) Schematic diagram depicts the SHIP2 consensus motif for the Mena and VASP EVH1 domains and corresponding sequences of the other known interaction partners of Mena and VASP (Zyxin, Lamellipodin, and Vinculin). (B) SHIP2 was immunoprecipitated from MDA-MB-231 cell lysates with either mouse (M) or rabbit (R) SHIP2 antibodies, or respective IgG controls, and immunoprecipitates were separated by SDS-PAGE and probed for Mena and VASP. (C) Coimmunoprecipitation was performed from MDA-MB-231 cell lysates with a mouse VASP antibody or mouse IgG control, and immunoprecipitates were probed for SHIP2, Mena, and VASP. (D) Coimmunoprecipitation was performed from MDA-MB-231 cell lysates expressing GFP-SHIP2 using a rabbit Mena antibody or rabbit IgG control, and immunoprecipitates were probed for SHIP2, Mena, and GFP. (E) HEK293 cells were transiently cotransfected with HA-SHIP2 or HA-SHIP2ΔFP4 and GFP-VASP as indicated, SHIP2 was immunoprecipitated from the cell lysates with an anti-HA antibody, and immunoprecipitates were probed for the presence of GFP-VASP. (F) As in D, HA-SHIP2 or HA-SHIP2ΔFP4 and GFP-Mena were cotransfected as indicated and immunoprecipitated with an anti-HA antibody, and immunoprecipitates were probed for the presence of GFP-Mena. (G) HEK293 cells were transiently cotransfected with HA-SHIP2 and mutants of GFP-VASP (WT, VASPΔCC, VASPΔEVH1, and VASPΔCC and ΔEVH1) as indicated, SHIP2 was immunoprecipitated from the cell lysates with anti-HA antibody, and immunoprecipitates were probed for the presence of GFP-VASP. (H) As in G, HA-SHIP2 and mutants of GFP-Mena (WT, MenaΔCC, MenaΔEVH1, and MenaΔCC and ΔEVH1) were cotransfected as indicated and immunoprecipitated with an anti-HA antibody, and immunoprecipitates were probed for the presence of GFP-Mena. (I) GST, GST-VASP-EVH1, and GST-Mena-EVH1 were coupled to glutathione-Sepharose beads and used to pull down interaction partners from the lysates of MDA-MB-231 cells. Resulting samples were probed for Vinculin and SHIP2 as indicated.

SHIP2 interacts with the EVH1 domains of Mena and VASP via the FPPPP motif. (A) Schematic diagram depicts the SHIP2 consensus motif for the Mena and VASP EVH1 domains and corresponding sequences of the other known interaction partners of Mena and VASP (Zyxin, Lamellipodin, and Vinculin). (B) SHIP2 was immunoprecipitated from MDA-MB-231 cell lysates with either mouse (M) or rabbit (R) SHIP2 antibodies, or respective IgG controls, and immunoprecipitates were separated by SDS-PAGE and probed for Mena and VASP. (C) Coimmunoprecipitation was performed from MDA-MB-231 cell lysates with a mouse VASP antibody or mouse IgG control, and immunoprecipitates were probed for SHIP2, Mena, and VASP. (D) Coimmunoprecipitation was performed from MDA-MB-231 cell lysates expressing GFP-SHIP2 using a rabbit Mena antibody or rabbit IgG control, and immunoprecipitates were probed for SHIP2, Mena, and GFP. (E) HEK293 cells were transiently cotransfected with HA-SHIP2 or HA-SHIP2ΔFP4 and GFP-VASP as indicated, SHIP2 was immunoprecipitated from the cell lysates with an anti-HA antibody, and immunoprecipitates were probed for the presence of GFP-VASP. (F) As in D, HA-SHIP2 or HA-SHIP2ΔFP4 and GFP-Mena were cotransfected as indicated and immunoprecipitated with an anti-HA antibody, and immunoprecipitates were probed for the presence of GFP-Mena. (G) HEK293 cells were transiently cotransfected with HA-SHIP2 and mutants of GFP-VASP (WT, VASPΔCC, VASPΔEVH1, and VASPΔCC and ΔEVH1) as indicated, SHIP2 was immunoprecipitated from the cell lysates with anti-HA antibody, and immunoprecipitates were probed for the presence of GFP-VASP. (H) As in G, HA-SHIP2 and mutants of GFP-Mena (WT, MenaΔCC, MenaΔEVH1, and MenaΔCC and ΔEVH1) were cotransfected as indicated and immunoprecipitated with an anti-HA antibody, and immunoprecipitates were probed for the presence of GFP-Mena. (I) GST, GST-VASP-EVH1, and GST-Mena-EVH1 were coupled to glutathione-Sepharose beads and used to pull down interaction partners from the lysates of MDA-MB-231 cells. Resulting samples were probed for Vinculin and SHIP2 as indicated.

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