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. 2006 Apr 20;116(5):1337–1345. doi: 10.1172/JCI27400

Figure 6. CD2AP SH3 domains interact directly with Synpo, but CD2AP does not interact with Neph1.

Figure 6

(A) FLAG-tagged Synpo (Synpo-long) was coexpressed with myc-tagged proteins, including full-length CD2AP (lane 1, CD2AP FL), CD2AP lacking the SH3 domains (lane 2, ΔSH3 domains), and CD2AP lacking the proline-rich domains (lane 3, ΔPR domains). Anti-FLAG immunoprecipitates were immunoblotted with an anti-myc antibody (upper panel). The expression of wild-type and mutated CD2AP proteins (input lysate; anti-myc) and Synpo is shown in the middle and lower panels, respectively. (B) FLAG-tagged Synpo (lane 1, FLAG–Synpo-long) and FLAG-tagged GFP (lane 2) were overexpressed, and anti-FLAG immunoprecipitates were blotted with recombinant, biotinylated GST-CD2AP fusion protein containing the 3 SH3 domains (left panel) or immunoblotted with anti-FLAG antibody (right panel). (C) FLAG-tagged GFP (lane 1, control), FLAG-tagged Synpo-long (lane 2), FLAG-tagged Synpo-short (lane 3), and FLAG-tagged Synpo-T (lane 4) were coexpressed with myc-tagged full-length CD2AP. Immunoprecipitates were prepared with an anti-FLAG antibody and immunoblotted with an anti-myc antibody (upper panel). (D) Neph1–yellow fluorescent protein (Neph1-YFP) was coexpressed with myc-tagged full-length CD2AP (lane 1) or myc-tagged nephrin (lane 2). Anti-myc immunoprecipitates were immunoblotted with anti-YFP antibody (upper panel). The expression of Neph1-YFP input lysate and myc-tagged nephrin or CD2AP controls is shown in the middle and lower panels, respectively. (E) Neph1-YFP (lane 1) and myc-nephrin (lane 2) were coexpressed with full-length FLAG-tagged CD2AP. Immunoprecipitates of FLAG-CD2AP were immunoblotted with anti-myc and anti-YFP antibodies. Neph1-YFP or myc-nephrin input lysates (anti-myc, anti-YFP) and CD2AP expression are demonstrated in the middle and lower panels, respectively.