To determine the involvement of phosphovimentin in the density of SERT on platelet plasma membrane, platelets in PRP were first pretreated with 5HT for 30 min at RT, then the pelleted platelets was biotinylated with membrane impermeable NHS-SS-biotin. Biotinylated platelet plasma membrane proteins were retrieved on streptavidin beads and eluted from the beads. Half of each biotinylated sample was subjected to immunoblot analysis using anti-SERT Ab. The biphasic effect of plasma 5HT on the density of SERT on platelet plasma membrane was observed, as seen previously in hypertension model systems. An intermediate level 5HT-stimulation increased the density of SERT on the platelet; however, at high level, 5HT-stimulation lowered the surface density of SERT compared to untreated platelets. Here, our data demonstrate that the association between SERT and vimentin is altered in a 5HT-dependent manner. Therefore, here we tested whether the cellular distribution of SERT is altered by 5HT-dependent phosphorylation of vimentin. The level of phosphovimentin on the plasma membrane 5HT-stimulated platelet was evaluated. The other half of the biotinylated platelet plasma membrane proteins was subjected to immunoblot analysis with pS56-Ab. Phosphovimentin appeared as one of the proteins associated with biotinylated plasma membrane-bound proteins in 5HT-stimulated platelets. SERT could be one of the other phosphovimentin-associated membrane proteins, but our co-IP data in 5HT-stimulated platelets also demonstrated an elevation in the association of SERTphosphovimentin in whole platelet. Therefore, we tested SERT-phosphovimentin association in 5HT-stimulated platelets. The effects of 5HT-stimulation on the amount of intracellular SERT mirrored those of the cell surface SERT. Previously, it has been shown that 5HT-stimulation phosphorylates vimentin on the Serine56 residue, but the vimentin S56A mutant is not phosphorylated by 5HT-stimulation. Therefore, to mechanistically determine how the vimentin-SERT association responds to 5HT for regulating the distribution of transporter molecules between plasma membrane and intracellular locations, the S56A mutant and the C-terminus Benzethonium Chloride truncated forms of SERT were Amikacin hydrate studied in a CHO heterologous expression system. Obviously, not all aspects of 5HTbiology in platelets can be recapitulated in CHO cells, but the CHO model system allows for the analysis of the association between vimentin and the C-terminus truncated forms of SERT and the nonphosphorylated mutant form of vimentin S56A. To ascertain the optimal 5HT concentration required to stimulate CHO cells expressing hSERT, we measured the density of SERT proteins on the plasma membrane of CHO-hSERT cells and compared this finding to human platelet membranes using biotinylation. In this previous study, we tried to model the effect of plasma 5HT on platelet SERT in a heterologous expression system. Simply stated, an equal amount of biotinylated membrane proteins from CHO-SERT cells and platelets were resolved and analyzed by W/B using SERT-Ab. These calculations together with the dose response analysis of CHO cells to 5HT-stimulation which was already conducted in our previous studies showed that the expression of SERT on the plasma membrane of CHO-SERT cells was 59-fold higher than on the platelet membrane. This estimation indicated that the effect of plasma 5HT at a concentration of 1 nM on platelet SERT may correspond to exogenous 5HT at a concentration of,45 mM on CHO-SERT cells. The co-localization of the red vimentin and green YFP-SERT signals were captured in the overlaid images with YFP and Texas Red filter sets.