Figure 1. SERT and vimentin interaction in human platelets.

(A) Endogenous vimentin and SERT expression in platelets were analyzed with W/B analysis. The association between vimentin and SERT was determined by a co-IP in platelets stimulated with 0, 1, and 2 nM 5HT. Following 5HT-stimulation, platelet lysate was divided into two portions; the half portion of lysate was incubated in anti-vimentin monoclonal Ab (B), the other half portion in anti-SERT Ab coated protein A Sepharose beads (C). Next day, vimentin- or SERT-Ab pulled down proteins were eluted from sepharose beads; both IP eluents were analyzed either with a polyclonal SERT Ab, or with pS56-Ab, respectively. Nonspecific adsorption of Sepharose beads was not determined in the absence of antibodies. The association between endogenous vimentin and SERT was altered in a 5HT concentration-dependent manner. The highest amount of SERT was pulled down by vimentin-Ab in 2 nM 5HT-stimulated platelets. Additionally, vimentin associated with SERT after 2 nM-5HT stimulation was in phosphorylated form. (D) Expression of SERT and vimentin in total cell lysates was determined by W/B analysis as a loading control. All lanes contain protein recovered from the same number of platelets (1.5×10⁸). Figures show representative images from 2 to 4 separate experiments.