FIGURE 3.

Requirement for external calcium in the Epo-stimulated calcium increase in HEK 293T cells. Fura Red-loaded HEK 293T cells were transfected with BFP-TRPC3 and Epo-R. A, cells were treated with 40 units/ml Epo in the presence (0.68 mm) or absence (2 mm EGTA) of extracellular calcium. B, cells were treated with or without 40 units/ml Epo in the presence of 2 mm EGTA, and 3 mm CaCl₂ was added at 10 min. [Ca²⁺]_i was measured at 2–5-min intervals for 20 min, and the peak percentage increase of [Ca²⁺]_i above base line was calculated for each cell. Mean ± S.E. of the peak percentage increase in [Ca²⁺]_i at different time points is shown. 21–38 individual cells were studied for each condition in two experiments. *, a significant increase in [Ca²⁺]_i compared with cells treated for 20 min with PBS (p < 0.0001).