Figure 1.

Validation of the existence of cytosolic and membrane-associated pools of calnuc. (A) Release of LDH from permeabilized cells. NRK cells were permeabilized with the indicated concentrations of digitonin, and supernatant was collected for assessment of LDH activity. LDH release increases linearly with increasing concentrations of digitonin. Results (mean ± SD) represent the average of values obtained in three separate experiments performed in triplicate. (B) Release of cytosolic calnuc after digitonin treatment. NRK cells were permeabilized with digitonin as in A. Released calnuc was precipitated from the supernatant with 10% trichloroacetic acid, separated by SDS/PAGE, and immunoblotted with affinity-purified anti-calnuc IgG. Increasing amounts of calnuc are released with increasing digitonin concentrations. (C) Pulse–chase experiments. NRK cells were pulse-labeled with [³⁵S]Met (300 μCi/dish) for 5 min and chased for the indicated time intervals. [³⁵S]calnuc was immunoprecipitated from cytosolic (S) and membrane (P) fractions, followed by autoradiographic analysis. The membrane-associated pool and the cytosolic pool have the same apparent mobility at the beginning of the chase (0), whereas after 5 and 10 min of chase, the membrane pool runs faster than the cytosolic pool. After 30 min of chase, the membrane pool again has approximately the same mobility as the cytosolic pool. Experiments were repeated three times.