FIGURE 4.

Evidence for functional CSR and Ca²⁺ signaling in SMIE cells. A and B, mean fluorescence traces of Fluo-4-loaded SMIE cells. Cells bathed in 0.5 mm Ca²⁺ were treated with 10 mm l-Phe or l-Phe (10 mm) plus 1 μm NPS-2143 (A). l-Phe with 0.5 mm external Ca²⁺ was added to control or shTRPC3-transfected cells (B). Ca²⁺ (2 mm) addition is shown on the trace. B, inset, Western blots (IB) using anti-TRPC3 antibody, showing knockdown of TRPC3 protein by TRPC3 shRNA (31). β-Actin was used as a loading control for TRPC3 protein. C, mean fluorescence traces of Fura-2-loaded SMIE cells. Cells were transfected with scrambled (control) or siCSR. Both groups of cell were bathed in buffer with 0.5 mm external Ca²⁺ and then treated with 10 mm l-Phe. C, inset, Western blots using anti-CSR antibody showing knockdown of CSR protein by siRNA to CSR. β-Actin in the lower panel represents as loading control for CSR protein. Additions/removals of Ca²⁺ in the external medium are indicated in the figure.