Figure 4: Yes1-mediated regulation of OCT2 tyrosine phosphorylation.

(a) Schematic representation of Yes1 protein (upper panel) showing the SH3 domain. The lower panel shows the putative proline-rich SH3 binding sequence in OCT2. (b) Endogenous Yes1 was immunoprecipitated from Hela-Vector and Hela-OCT2 cell lysates using a mouse anti-Yes1 antibody, followed by western blot analysis with rabbit anti-FLAG and Yes1 antibodies. (c) Plasmids for OCT2 mutants were transiently transfected into Hela cells and 24 h later, uptake assays (15 min) were performed using [¹⁴C]-TEA (2 μM) or [¹⁴C]-metformin (50 μM). The uptake levels were normalized to protein concentration in each group. The graph represents relative OCT2 function (TEA or metformin uptake) as compared to wild-type OCT2 transfected group. * indicates statistically significant as compared with wild-type group (P<0.05, Student’s t-test). (d) Hela cells were transiently transfected with indicated FLAG-tagged OCT2 constructs, followed by immunopreicipation with anti-FLAG antibodies and western blot analysis by FLAG and phosphotyrosine antibodies. (e) Proposed model of Yes1 and OCT2 interaction. (f) Plasmids for OCT1 and OCT3 mutants were transiently transfected in Hela cells and 24 h later, uptake assays (15 min) were performed using [¹⁴C]-TEA (2 μM). The uptake levels were normalized to protein concentration and the graph represents relative OCT2 function (TEA uptake) as compared with respective wild-type group. * indicates statistically significant as compared to wild-type group (P<0.05, Student’s t-test). All experimental values are presented as mean±s.e.m. The height of error bar=1 s.e.