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. 2017 Apr 12;37(15):4046–4064. doi: 10.1523/JNEUROSCI.3319-16.2017

Figure 6.

Figure 6.

Interaction of Rap2-Tiam2/STEF activates the Rac1 GEF activity of Tiam2/STEF and these signaling proteins colocalize with flotillin1 via GPM6a. A, Rap2-binding sites in Tiam2/STEF. Top, HA-tagged mutants of Tiam2/STEF used for the Rap2 binding assay. Bottom, Determination of the Rap2-binding domain of Tiam2/STEF for Rac1 activation. Full-length or deletion mutants of HA-STEF were coexpressed with Flag-Rap2 in COS-7 cells. Cell lysates were immunoprecipitated with anti-HA antibody, followed by immunoblotting with anti-Flag and anti-HA antibodies. Flag-Rap2 that coprecipitated with HA-STEF is indicated by an arrow. B, GTP-Rap2 activated the Rac1-GEF activity of Tiam2/STEF. Myc-tagged Rac1 was preincubated with either DN (S17) or CA (V12) Rap2 and HA-STEF. Activated Rac1 was pulled down using PAK-PBD immobilized to beads and was analyzed using Western blotting (arrow). C, Representative triple immunostaining of the localization of endogenous flotillin1 (green), Rap2 (blue), and Tiam2/STEF (red) in WT or GPM6aKO cortical neurons. nMDP values (see also Fig. 7D and Materials and Methods) indicate that flotillin1 colocalized with Rap2 or Tiam2/STEF in the WT neuron, but not in the GPM6aKO neuron. Scale bar, 10 μm.