Fig. 6. Colocalization of NHERF with the 5-HT_4(a)R, but not the 5-HT_4(b)R splice variant in transiently transfected NIH-3T3 cells. (A) Cells were transfected by electroporation with constructs encoding HA-tagged NHERF (1), the Rho-tagged 5-HT_4(a)R (2) or the Rho-tagged 5-HT_4(b)R (3) alone. They were permeabilized, immunostained with the anti-HA (1:400) or the anti-RhoTag (1:100) antibody and analysed by confocal laser scanning microscopy. Mid-nuclear slices from representative cells are shown. (B,C) Cells were transfected with either the HA-NHERF-RhoTag 5-HT_4(a)R/pIRES2 (B) or the HA-NHERF-RhoTag-5-HT_4(b)R/pIRES2 (C) construct, which ensure efficient co-expression in the same cells. Confocal slices are shown from the mid-nuclear level (top) or close to the cell surface (bottom). Left, medium and right panels show HA staining, RhoTag staining and merged images, respectively. Bars, 10 µm.