FIGURE 4.

NK1R activation regulates NET/NK1R association and subcellular compartmentalization. HTR cells transfected with His-hNET and HA-hNK1R were treated with 0.25 μm SP for the indicated times and subjected to (A) cell surface biotinylation and isolation of avidin-bound (surface) and unbound (intracellular) fractions followed by (B) hNET immunoprecipitations (IP) as described under “Experimental Procedures.” A, biotinylation: representative blots from three independent biotinylation experiments show total (T), surface (avidin B), and intracellular (avidin UB) levels of hNET and NK1R. Note the time-dependent decreases in the surface levels of hNET and NK1R proteins with concomitant increases in the intracellular pools. B, immunoprecipitation: representative immunoblots from three independent experiments show co-immunoprecipitation of NK1R from surface and intracellular hNET. Note the robust NK1R association with surface hNET at 0 time/vehicle, which disappeared following SP treatment. In contrast, note the very little NK1R association with intracellular hNET at 0 time/vehicle that increased time dependently following SP treatment.