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. Author manuscript; available in PMC: 2008 Dec 13.
Published in final edited form as: Cell. 2008 Jun 13;133(6):963–977. doi: 10.1016/j.cell.2008.04.039

Figure 7. VAP MSP domains bind to Eph receptor extracellular domains.

Figure 7

(A) Diagram of the C. elegans proximal gonad. MSP binds to receptors on the oocyte (Oo) and sheath cell surfaces in the proximal gonad (Miller et al. 2003; Corrigan et al. 2005).

(B) Receptor binding sites are visualized using FITC-conjugated MSP domains. FITC-conjugates are biological active in promoting oocyte maturation and sheath contraction (Miller et al. 2003; data not shown). Compete includes a 25-fold molar excess of unlabelled protein. Bar, 20 μm.

(C, D) VPR-1 MSP domain can bind to VAB-1 Eph receptor. Purified FLAG-tagged VPR-1 MSP (VPR-1MSP-FLAG) and V5 tagged VAB-1 ECT domain (VAB-1 Ex-V5) were co-immunoprecipitated using FLAG antibody (C) and V5 antibody (D). IP, immunoprecipitation.

(E) His- and V5- tagged mouse EphA4 ectodomain (mEphA4Ex-V5His) and a His tagged hVAPB MSP (hVAPMSP-His) co-immunoprecipitate using anti-V5 antibody.

(F) Conditioned medium containing hVAPMSP disrupts the interaction between mouse EphrinB2 (mEphrinB2) and EphA4. WCL: whole cell lysates.

(G) Quantification of the fraction of IP of (F). Lanes 1 to 4 correspond to the lanes in Figure 7F.

(H) Ephrin B2 competes with hVAPB MSP for binding of EphA4 in a dose dependent manner. As decreasing amounts of EphrinB2 are added, increasing amounts of hVAP MSP are pulled down by mEphA4Ex-V5 His. Note that EphrinB2-His cannot be detected in the top panel because it has a similar molecular weight to the IgG light chain. * shows non-specific heavy and light chains of IgG.

(I) The quantification of the fraction of IP of (H). The lanes correspond to the lanes 1 to 4 in Figure 7H.