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. 1996 Dec 10;93(25):14574–14579. doi: 10.1073/pnas.93.25.14574

Figure 2.

Figure 2

Calcium-dependent calmodulin stimulation of active nuclear import in vitro. HeLa cells were permeabilized with digitonin and assayed for nuclear transport in the absence of rabbit reticulocyte lysate, but in the presence of 30 μM calmodulin (Calbiochem, high purity) and 9.2 μM calcium (A). Incubations were also carried out in the absence of creatine kinase, creatine phosphate, or ATP, but in the presence of 6 units/μl hexokinase and 6 mM glucose (B), in the presence of 0.4 mg/ml wheat germ agglutinin (C), at 4°C (D), with B-phycoerythrin bearing an altered targeting peptide that is not sufficient to support transport to the nucleus (ref. 17, E), or in the absence of added calmodulin (F). Nuclear import was assayed at varying calcium concentrations (G) in the presence of 30 μM calmodulin and ATP in the absence (○, broken line) or presence (•, solid line) of 1 mM GTP, or in the presence of 5 mM GTPγS (□). Data represent the mean ± SD (not included for assays in the presence of GTPγS) of two independent experiments. Images in E and F were obtained with an 100× oil immersion objective; all other images were collected with a 63× objective.