FIGURE 7.

Activation of CSR in SMIE cells enhanced CSR-TRPC3 complex formation and plasma membrane expression of TRPC3. A, SMIE cells grown in Transwell filter treated with l-Phe and/or NPS-2143 were solubilized using RIPA buffer, and co-immunoprecipitation (IP) experiment was performed using anti-CSR antibody (1:100). i, Western blot using anti-TRPC3 antibody (1:500) shows changes in TRPC3 bands representing the increase in complex formation induced by the treatment with l-Phe. ii, complex formation is inhibited after SMIE cells are treated with l-Phe (10 mm) and/or NPS-2143 (1 μm). B, cell surface biotinylation experiment in SMIE cells was performed to determine plasma membrane expression of TRPC3 in response to CSR activation. SMIE cells grown in Transwell filter were treated with l-Phe (10 mm) and/or NPS-2143 (1 μm) then biotinylated using EZ-Link Sulfo-NHSSS-Biotin and solubilized using RIPA buffer. Co-IP experiments were performed using NeutrAvidin beads followed by Western blotting using anti-TRPC3 (1:500) and Na⁺,K⁺-ATPase (NaK; 1:1000) antibodies showing changes in TRPC3 bands representing an increase in surface expression induced by l-Phe (i). More importantly, treatment with l-Phe and/or NPS-2143 inhibited the complex formation (ii). Another plasma membrane protein did not change, indicating the specificity of the biotinylation experiment (lower panel).