Figure 2. Effect of 5HT stimulation on SERT-vimentin association on platelet plasma membrane.

0–2 nM 5HT-stimulated platelets were biotinylated with NHS-SS-biotin [15], [16], [19], and biotinylated plasma membrane proteins were retrieved on streptavidin beads. Half of biotinylated platelet membrane proteins were analyzed for SERT (A), and the other half was analyzed for anti-phosphovimentin-Ab (pS56-Ab) (B) with W/B. Neither SERT not pS56-Ab detected recognizable proteins in mock biotinylated controls (carried through the biotinylation procedure without the addition of the biotinylation reagent); therefore, they are not presented in the blots. Plasma 5HT levels had a biphasic effect on the plasma membrane expression of platelet SERT [16]. To correlate SERT-phosphovimentin association on the surface density of platelet SERT, the biotinylated platelet membrane proteins were analyzed with pS56-Ab. Plasma 5HT, only at high level, showed an association between phosphovimentin-SERT. Intracellular SERT (C) and vimentin (D) were determined by W/B analysis of non-bound material and also served as a loading control. All lanes contain protein recovered from the same number of platelets (1.5×10⁸). Figures show representative images from 3 separate experiments.