Fig. 1. Description of the co-culture system with analysis of secreted contactins.
(A) Schematic structure of a contaction showing the six immunoglobulin-like domains and the four fibronectin-type III domains. The protein is attached to the membrane by a GPI moiety, and a soluble form is secreted upon GPI hydrolysis. (B) Membrane staining by immunofluorescence of non-permeabilized HEK293 cells after transfection with the Cntn4, -5 or -6 cDNA. Fixed cells were incubated with primary antibodies, then with Alexa 488-conjugated (Cntn4) and Alexa 594-conjugated (Cntn5 and -6) antibodies to Cntn as described in Materials and Methods. Scale bars = 20 µm. (C) Semi-quantitative analysis of fluorescence in permeabilized HEK293 cells. Number of cells analyzed is indicated in the graphs. Values are the mean±s.e.m. Statistical analysis was performed using the Student's t-test. No significant difference was observed (P>0.05). (D) MAP2 staining of primary cortical neurons cultured in the presence of transfected HEK293 cells. In blue: DAPI nuclear staining. Some representative neurons are depicted in the absence and in the presence of Cntn4, Cntn5 or Cntn6. Note a higher ramification of neurons in the presence of Cntn5 as compared to Cntn4 and -6. Scale bars = 20 µm. (E) Representative western blot of culture supernatants and HEK293 cell extracts for the detection of Cntn4, -5, and -6 proteins. Controls were obtained from HEK293 cells transfected with the empty vector (EV). Two cell densities (C1, C2) corresponding respectively to ∼1.5×105 and 3×105 cells were tested. (F) Representative western blot of co-culture supernatants. Rat cortical neurons were cultured in the presence of transfected HEK293 cells for 48–72 hours. No contactin proteins were detectable in the absence of HEK293 cells. Duplicates correspond to two independent co-cultures. (G) Western blot analysis of 0.5 to 25 ng of mouse Cntn6 and one aliquot of a co-culture supernatant for Cntn6. Exposure times were the same between all samples.