Figure 7: MAP6 protein is involved in the Sema3E signalling pathway.
(a) Immunoprecipitation of MAP6-E-GFP and Sema3E receptor proteins. HEK 293T17 cells were transfected with MAP6–E-GFP, VEGFR2, Plexin D1 or Neuropilin1 cDNAs. Immunoprecipitation was performed using a polyclonal anti-PlxD1 antibody. In control experiments, Plexin D1 cDNA was omitted. (b) Immunoprecipitation of endogenous MAP6-E from subicular neurons using an anti-MAP6 23N antibody. (c) Rescue experiments on MAP6-KO neurons by MAP6-E-GFP constructs and Sema3E response. Schematic representation of MAP6-E-GFP, MAP6-E-ΔMnΔMc-GFP, MAP6-E-ΔMc-GFP, MAP6-E-ΔMn-GFP and MAP6-E-ΔPRD39-57-GFP constructs. Values correspond to mean axonal length and were normalized to 100% for values obtained in control conditions (GFP without Sema3E). All quantifications have been repeated at least three times using independent neuronal cultures yielding consistent results (number of neurons≥70 in all conditions). Error bars, s.e.m. **P<0.01; ***P<0.001 Student’s t-test. (d) Immunoblot analysis of synaptosomal proteins eluted from glutathione-Sepharose columns containing GST or GST fused to SH3 domains from intersectin (ITSN1/SH3A-E domains), PLC-γ1, cSrc, and p85/PI3K. The blot was stained with Ponceau red and probed with 23N antibody. (e) Immunoblot analysis of postnatal brain proteins eluted from glutathione-Sepharose columns containing GST and GST-MAP6 LNt fragment containing the PRD39-57 domain. Blots were stained with Ponceau Red and probed with anti-p85 or anti-intersectin antibodies. (f) Immunolabelling, using anti-phospho-Akt antibodies, of subicular neurons treated 15 min with 10 nM of Sema3E. Histogram shows the relative immunofluorescence signal of phospho-Akt staining within axons and growth cones (number of neurons≥60). Error bars, s.e.m. ***P<0.001.
