Binding of TRPC6 to SNF8 in 293T cells. A. M1R cells expressing FLAG-tagged TRPC6 under a tetracycline inducible promoter were transfected with HA-tagged SNF8 or control vector, followed by culture in the absence or presence of 1 μg/ml tetracycline to induce the expression of TRPC6. Cell lysates were either directly analyzed for TRPC6 and SNF8 expression by Western blot (top and middle panel, respectively), or incubated with FLAG M2 affinity beads to immunoprecipitate TRPC6. HA-tagged SNF8 was detected bound to the affinity beads when expressed in the presence of TRPC6 (bottom panel). B. Cells transfected with HA-tagged SNF8 and induced to express TRPC6 as indicated were treated with or without 100 μM carbachol for 5 minutes prior to lysis and immunoprecipitation as in (A) above.