RBCs express functional TRPC1. (A) RBC CR1 ligation promotes a SAC-dependent Ca⁺⁺ influx. RBCs were loaded with Fluo-4 and incubated with buffer, 1 or 5μM GsMTX-4 for 30 minutes and then analyzed by flow cytometry for CR1-mediated Ca⁺⁺ influx (arrow) as described above. (B) RBCs express TRPC1. RBCs were fixed, permeabilized, and incubated with either control (left panel) or rabbit monoclonal anti-TRPC1 (right panel) for 16 hours, washed, and incubated with goat anti–rabbit AlexaFluor488. Cells were imaged under the microscope using the fluorescein isothiocyanate/green fluorescent protein filter. (C) Immunoblotting detection of TRPC1 in RBCs. Testes tissue lysate (Santa Cruz Biotechnology; second lane) and RBC lysate (third lane) were separated by electrophoresis using Tris-HCl gels and probed with rabbit anti-TRPC1 Ab (Santa Cruz Biotechnology). TRPC1 is seen as a band at approximately 87 kDa (arrow) in both positive control and RBC lysate. Lane 1, Bio-Rad molecular weight markers. (D) Anti-TRPC1 inhibitory antibody T1E3 binds RBCs. RBC were incubated with either 1/500 dilution of normal rabbit serum (dotted histogram) or T1E3 Ab anti-serum (continuous histogram) for 10 minutes followed by AlexaFluor488 goat anti–rabbit. Cells were washed and examined by flow cytometry. (E) CR1 ligation triggers a Ca⁺⁺ influx dependent on TRPC1. Fluo-4 loaded RBCs were preincubated with control serum or T1E3 for 20 minutes, washed twice, and then analyzed by flow cytometry for CR1-mediated Ca⁺⁺ influx (arrow) as described above.