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Fig. 3 Phosphatase activity of Xenotoca eiseni voltage-sensing phosphatase (Xe-VSP). A: the pulse protocol for estimating VSP activity consisted of a 50-ms hyperpolarizing pulse to −120 mV (test pulse to measure Kir2.1 currents) followed by a 300-ms depolarizing pulse to 100 mV (to induce VSP enzyme activity). The holding potential was −60 mV. This protocol was repeated 21 cycles without an interval (20). B: representative traces of Kir2.1 currents recorded from HEK193T cells coexpressing Xe-VSP (left) and without Xe-VSP (right) in whole cell patch configuration. All 21 current traces were superimposed, with the first stimulation highlighted in red. The traces in this figure focus on Kir2.1 currents during the hyperpolarizing test pulse. Full traces are available in Supplemental Fig. S3. C: time-dependent changes in normalized Kir2.1 inward currents in the presence and absence of Xe-VSP. Unfilled circles, data with Xe-VSP (n = 8). Filled circles, data without Xe-VSP (n = 5). Values were measured at the end of the 50-ms hyperpolarizing pulses and were normalized by the current amplitude during the first stimulation. Data are means ± SE. D: comparison of normalized Kir2.1 inward currents in the presence and absence of Xe-VSP during the final stimulation (same data set as in C). Data are means ± SE. ***P = 0.0004; unpaired Student’s t test. E: phosphatase activities of Xe-VSP, estimated as rate constants for the decrease in normalized Kir2.1 currents with single exponential fitting. Fitting was applied to the dataset from each cell (n = 8). Data are means ± SE. Note that the y-axis is calibrated in log scale.