FIGURE 4.

Effect of UCP3 knockdown on ER Ca²⁺ refilling in intact and permeabilized cells. A, SERCA2 immunoblot of HeLa cells transfected with Ctrl or UCP3 siRNA for 48 h. 50 μg/lane of protein from cell extracts was analyzed, using actin as loading control. B, left, averaged [Ca²⁺]_ER recordings of intact HeLa cells during ER Ca²⁺ refilling. After 15 μm BHQ induced Ca²⁺ release in Ca²⁺-free medium, cells were washed, and Ca²⁺ was then added to assess the kinetics of store refilling. Data were fitted with the sigmoidal equation to extract the EC₅₀ (right), and they are mean ± S.E. of 40 (n = 5) and 30 cells (n = 4) for Ctrl and UCP3 siRNA, respectively. **, p < 0.01. C, left, averaged [Ca²⁺]_ER recordings during ER Ca²⁺ release and refilling in permeabilized cells. The K⁺-rich intracellular buffer was supplemented with 1 mm Mg-ATP and 1 mm MgCl₂ to allow ER Ca²⁺ refilling. Where indicated, 100 μm digitonin (dig), 15 μm BHQ, and 100 nm free Ca²⁺ were added. Right, statistical evaluation of the ER Ca²⁺ refilling kinetics. EC₅₀ was determined as described for B. Data are mean ± S.E. of 47 (n = 3) and 33 cells (n = 3) for Ctrl and UCP3 siRNA, respectively. NS, not stimulated.