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. 2011 Apr 20;31(16):6174–6187. doi: 10.1523/JNEUROSCI.5464-10.2011

Figure 8.

Figure 8.

Downregulation of Nrp and PlexinA in MGE cells derived from Robo1−/−;GAD67-GFP mice and Robo1-DN-transfected GN11 cells. A, B, Sema3A and Sema3F do not bind to Robo1. Cell lysates from COS-7 cells, which had been transiently transfected with myc-tagged full-length Plexin, Nrp, or Robo1 and treated with Flag-tagged Sema3F in the culture medium, were immunoprecipitated with anti-myc antibody and immunoblotted with anti-Flag and anti-myc antibodies (A). B, Sema3A-AP or Sema3F-AP was added to COS-7 cells transiently transfected with full-length Nrp1, Nrp2, Robo1, or control. Sema3A binding was only observed with cells expressing Nrp1, and Sema3F binding was only observed with cells expressing Nrp2; no binding was observed to cells expressing Robo1. C, QPCR for semaphorin receptors was performed on GN11 cells transfected with Robo1-DN or control (GFP) constructs and of MGE cells derived from E15.5 Robo1+/−;GAD67-GFP and Robo1−/−;GAD67-GFP mice. D, Immunoblot analyses of MGE cells derived from E15.5 Robo1+/−;GAD67-GFP and Robo1−/−;GAD67-GFP mice, and of GN11 cells transfected with Robo1-DN cells showed reduced levels of Nrp1 and PlexinA1, but not Nrp2. Abbreviations: −RT, Without reverse transcriptase (negative control). Genes examined are listed next to the gel bands. E, QPCR for VEGF receptors was performed on MGE-derived cells from E15.5 Robo1+/−;GAD67-GFP and Robo1−/−;GAD67-GFP mice. Genes examined are listed next to the gel bands. Lack of expression of VEGF receptors was observed in MGE cells derived from both groups of animals but were present in total brain extract.