Fig. 3

Effects of SEC5 on InsP₃R channel activity. a, b Representative Ca²⁺ transients in HEK293 cells stimulated by Ca²⁺-free Hank’s balanced salt solution (white bar), followed by addition of 50 μM carbachol to the extracellular solution (black bar). a Cells were transfected with pEGFP-SEC5 or pEGFP-N1 vectors. Representative western blot depicting the expression of SEC5 in cells transfected with pEGFP-SEC5 or pEGFP-N1 vectors. Actin was used as a loading control. b Cells were transfected with scramble siRNA or SEC5-siRNA. A western blot depicting the knock down of SEC5 by siRNA is shown. Data are summarized as the mean ± SEM from three experiments with more than 100 GFP-positive cells analyzed. c Co-immunoprecipitation assays in RAW264.7 lysates demonstrated that recombinant H1 peptide competed with endogenous InsP₃R3 for binding to SEC5. The bar chart summarizes the amount of InsP₃R3 co-immunoprecipitated by anti-SEC5 antibodies. Data represent the mean ± SEM intensities of the co-immunoprecipitated InsP₃R3 band from three experiments. d Representative Ca²⁺ traces depicting carbachol-induced ER Ca²⁺ release (black bar) under the influence of recombinant H1 peptide or H1 scramble peptide. Data are summarized as the mean ± SEM from three experiments with at least 100 cells analyzed. e Representative single-channel current traces activated by 500 nM InsP₃ and 2 μM free Ca²⁺ in the pipette solution. Arrow indicates zero-current level, whereas downward deflection represents channel openings at pipette voltage = 40 mV. The channel open probability was enhanced by the addition of SEC52 peptide in the pipette solution. Data represent the mean ± SEM for at least three individual nuclear patches. (*p < 0.05, **p < 0.005, ***p < 0.001)