Figure 6.

Most of the ⁴⁵Ca²⁺ taken up by CALNUC in the Golgi is released by the SERCA inhibitor Tg. Cells were incubated with ⁴⁵Ca²⁺ for 48 h as in Fig. 5 and sequentially treated with 0.1 μM Tg, 2 μM ionomycin, and 2 μM monensin. ⁴⁵Ca²⁺ release into the medium was measured after each treatment. (A) ∼73%, ∼20%, and 7% of the ⁴⁵Ca²⁺ was released from HeLa cells transiently overexpressing CALNUC-GFP after Tg, ionomycin, and monensin treatments, respectively. Nontransfected HeLa cells (N.T.) or those stably expressing GFP alone did not release as much ⁴⁵Ca²⁺ as HeLa cells overexpressing CALNUC-GFP. (B) Similar results (70%, 25%, and 5%) for Ca²⁺ release were obtained for induced (5 μm ponasterone A for 24 h) EcR-CHO-CALNUC cells following sequential treatment as above. Induced cells release twice as much ⁴⁵Ca²⁺ as noninduced cells. Results (mean ± SD) represent the average of values obtained in three separate experiments performed in duplicate.