NCAM and FGF activate distinct FGFR-mediated signaling pathways. (A) Cell lysates (5 mg) from HeLa cells stimulated for 10 min with FGF-2, FGL, or NCAM-Fc were immunoprecipitated (IP) with control IgG or antibodies against individual FGFR types (FGFR1–4) and immunoblotted for phosphotyrosine (top) and for the corresponding FGFR types (bottom). (B, top) Equal loading for phospho–PLC-γ was verified by immunoblotting for vinculin. (B–D) HeLa cells were stimulated for 10 min with FGF-2, FGL, or NCAM-Fc with or without a pretreatment with PD173074 (B) or PP1 (D). (C) Cells were transfected with dn-Ras or an empty vector before the stimulation (see Fig. S2 B for the expression of dn-Ras). Cell lysates were immunoblotted for phospho–PLC-γ, phospho–FRS-2α, phospho-Shc, phospho-Src, and phospho-Erk1/2 followed by immunoblotting for total PLC-γ, FRS-2α, Shc, Src, and Erk1/2 as indicated.