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. 2005 Apr 25;169(2):341–354. doi: 10.1083/jcb.200409127

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Copyright © 2005, The Rockefeller University Press

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Figure 8. — Cis and trans interactions between NCAM140 and PrP increase targeting of NCAM140 to lipid rafts and fyn activation in CHO cells. (A and B) Lipid rafts (A) or fyn immunoprecipitates from lipid rafts (B) from low density cultures of nontransfected CHO cells or CHO cells stably transfected with NCAM140, PrP, or cotransfected with NCAM140 and PrP were analyzed by immunoblotting (WB) with antibodies to NCAM, fyn protein, and Tyr416-phosphorylated fyn. Note that levels of NCAM140 and activated fyn are increased in lipid rafts from CHO cells cotransfected with NCAM140 and PrP, but not singly transfected cells. (C and D) Confluent cultures of nontransfected and NCAM140-transfected CHO cells were incubated with Fc or PrP-Fc. Lipid rafts (C) or fyn immunoprecipitates from lipid rafts (D) were analyzed by immunoblotting (WB) with antibodies to NCAM, fyn protein, and Tyr416-phosphorylated fyn. Note that levels of NCAM140 and activated fyn are increased after incubation with PrP-Fc in NCAM140-transfected cells. Slightly increased basal levels of activated fyn in control NCAM140 transfected cells compared with nontransfected cells are induced by trans interactions between NCAM molecules in these cultures (Niethammer et al., 2002). Immunolabeling for fyn (A and C) shows that lipid rafts were isolated with the same efficiency. Diagrams with quantitation of immunoblots (A–D) are shown below. Mean values ± SEM (n = 6) are shown. AU = arbitrary units. *, P < 0.05, paired t test.