Figure 5: OCT2 tyrosine phosphorylation and functional regulation in vivo.

(a) Wild-type FVB mice were injected with either vehicle or dasatinib (15 mg kg⁻¹, p.o.) and 30 min later, the mice were euthanized and the kidneys were collected. Kidney tissue lysates were then used to immunoprecipitate endogenous Oct2 followed by western blot analysis by phosphotyrosine and Oct2 antibodies. (b) Male FVB mice were injected with dasatinib (15 mg kg⁻¹) followed by pharmacokinetic analysis of dasatinib levels in the plasma. (c) Wild-type and Oct1/2^−/− mice were injected with either vehicle or dasatinib (15 mg kg⁻¹, p.o.) and 30 min later they were injected with 0.2 mg kg⁻¹ [¹⁴C]-TEA (i.v.), followed by plasma collection at 5 min. The graph represents plasma TEA levels from n=5 mice per group. * indicates statistically significant as compared with wild-type vehicle group (P<0.05, Student’s t-test). (d) Isolated renal tubules were coincubated with dasatinib in the presence of the OCT2 substrate ASP (30 min), and relative uptake was measured compared with control group. (e) Wild-type FVB mice were injected with either control or Yes1 siRNA by hydrodynamic tail-vein injection (25 μg in 0.5 ml of PBS). Three days later, the mice were injected i.v. with a 0.2 mg kg⁻¹ dose of [¹⁴C]-TEA, and plasma levels of TEA were measured at 5 min (n=5 mice per group). Kidneys were also collected to determine Yes1 knockdown. (f) Wild-type and Yes1^−/− mice (n=5) were injected with 0.2 mg kg⁻¹ [¹⁴C]-TEA (i.v.) and plasma levels of TEA were measured at 5 min. (g) Kidney tissue lysates from wild-type and Yes1^−/− were used to immunoprecipitate endogenous Oct2 followed by western blot analysis by phosphotyrosine and Oct2 antibodies. All experimental values are presented as mean±s.e.m. The height of error bar=1 s.e.