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. 2009 Feb 13;284(7):4567–4581. doi: 10.1074/jbc.M804734200

FIGURE 1.

FIGURE 1.

Endogenous expression of TRPC3 and TRPC6 in human hematopoietic cells. A, Western blotting was performed on lysates from U937 cells, UT-7 and TF-1 Epo-responsive cell lines, CD34+ cells, and day 10 and 14 BFU-E-derived erythroblasts. Equivalent amounts of protein were loaded in each lane. Immunoblotting with anti-TRPC3 antibody demonstrated increased expression of TRPC3 during erythroid differentiation, whereas blotting with anti-TRPC6 showed that TRPC6 expression decreased. Blots were probed with anti-actin antibody to compare loading of lanes. Blots were also probed with anti-Epo-R antibody. Representative results of three experiments are shown. B, densitometry was used to quantitate TRPC3 and TRPC6 bands from three experiments of lysates from CD34+ cells and day 10 and day 14 BFU-E-derived erythroblasts. The TRPC3/TRPC6 ratio was calculated and normalized to CD34+ cells to allow comparison between experiments. The mean normalized ratio ± S.E. is shown. The TRPC3/TRPC6 ratio was significantly less in CD34+ cells compared with day 10 (**, p ≤ 0.05) and day 14 erythroblasts (*, p ≤ 0.002). TRPC3/TRPC6 ratio was significantly greater in day 14 compared with day 10 cells (***, p ≤ 0.02). C, Western blotting was performed on lysates from day 7 and 10 BFU-E-derived erythroblasts with antibodies to TRPC3, TRPC6, Epo-R, and actin. Equivalent amounts of protein were loaded in each lane. Representative results of three experiments are shown. D, densitometry was used to quantitate TRPC3 and TRPC6 bands from three experiments with day 7 and 10 BFU-E-derived erythroblasts. The TRPC3/TRPC6 ratio was calculated, and the mean ratio ± S.E. normalized to day 7 cells is shown. The TRPC3/TRPC6 ratio was significantly less at day 7 compared with day 10 (*, p ≤ 0.01). E, [Ca2+]i was measured in day 7 (n = 30) and 10 (n = 33) BFU-E derived erythroblasts at base line and over 20 min of Epo stimulation. The mean % increase in [Ca2+]i above base line was calculated (% increase = peak [Ca2+]i divided by base line [Ca2+]i × 100%, -100% (baseline); *, p ≤ 0.001.