Figure 11

Co-immunoprecipitation experiments showing homophilic interaction of B3 mediated by the sema domain. COS-7 cells were co-transfected with various full-length and deletion constructs of B3. Cells transfected with a putative interaction partner and the corresponding/respective vector lacking an insert served as negative controls. Cells were lysed and immunoprecipitations (IP) were performed by various antibodies. Total lysates and IPs were analyzed by Western blot (WB) using antibodies as indicated in the figures. Bands marked with * represent antibodies precipitated by protein-A-agarose. (A) Human B3 constructs used for the IP experiments. (B) Cells were co-transfected with pSecTag2B/B3 encoding myc-tagged full-length B3 and pEGFP-N1/B3 encoding EGFP-tagged full-length B3. IP was performed with anti-myc antibody and shows homophilic interaction of B3. (C-E) Co-IP of three different B3 deletion mutants with anti-myc antibody against myc-tagged full-length B3, demonstrating homophilic interaction mediated through the sema domain. Cells were co-transfected with pSecTag2B/B3 and pFLAG/B3Δic encoding B3 missing the intracellular part (C), pcDNA3.1/B3ΔsemaΔic encoding a V5-tagged fragment of B3 missing the intracellular part and the sema domain (D), or pcDNA3.1/B3sema encoding the V5-tagged sema domain of B3 (E). (F) Cells were co-transfected with pcDNA3.1/B3sema encoding V5-tagged sema domain of B3 and with pcDNA3.1/B3semaHA encoding HA-tagged sema domain of B3. Co-IP was performed using anti-HA antibody and suggests that the sema domain is essential and sufficient for homophilic binding.