FIGURE 5.

NK1R activation translocates NET·NK1R complexes from non-raft fractions to raft fractions. HTR cells transfected with His-hNET and HA-hNK1R were treated with vehicle or SP (0. 25 μm) for 15 min. Following treatments, the cells were solubilized in MBS containing 0.1% Triton X-100 and subjected to sucrose gradient centrifugation as described under “Experimental Procedures.” A, lipid raft analysis of NET, NK1R, and flotillin-1: equal aliquots (40 μl) of sucrose density gradient fractions (2–10) were subjected to SDS-PAGE followed by immunoblotting with antibodies to hNET, NK1R, and flotillin-1. Representative immunoblots from three independent experiments are shown. B, analysis of hNET/NK1R/PKCα associations in rafts and non-rafts: equal volumes (500 μl) of raft and non-raft fractions were used to isolate NET immunoprecipitates as described above and the immunoisolates were subjected to SDS-PAGE followed by sequential immunoblotting with antibodies to hNET, NK1R, and PKCα. Representative immunoblots from three separate experiments are shown.