Characterization of the TRPC1 antibody Ank and expression of TRPC proteins in human platelets.

(A) A 50 μg microsomal preparation of nontransfected and hTRPC1-transfected QBI-293A cells (QBI) was probed for Western blot detection with Ank antibody (5 μg/mL). (B) QBI-293A cells overexpressing hTRPC1 were lysed, immunoprecipitated (IP) with Ank, and probed with anti-HA (0.5 μg/mL). (C) Lysates of resting platelets were probed with Ank (left panel) or immunoprecipitated with Ank and probed with the Ank antibody (right panel). (D) 50 μg nontransfected and mTRPC6-transfected QBI-293A cells (left panel) and 50 μg platelet mixed membranes (right panel) were probed with anti-TRPC6 (1/400). (E) 50 μg mTRPC4α and mTRPC5-transfected QBI-293A cells and 100 μg mixed membranes from platelets and the megakaryocytic cell lines DAMI and CHRF-288 were probed with anti-bTRPC4 (1/200). (F) 50 μg nontransfected and hTRPC3-transfected QBI-293A cells and 50 μg platelet mixed membranes were probed with anti-αTRPC3 (1/2000). Molecular size markers are noted in kilodaltons (kDa) and TRPC protein sizes indicated with arrows. All Western blots are representative of at least 3 distinct determinations.