FIGURE 1.

CaM-NLS-dependent nuclear import of SRY requires hsc70. Nuclear import of GFP-SRY-HMG-wild type (WT) and its mutant derivatives (see schematics in B depicting effects of the mutations) was reconstituted in vitro in the absence or presence of specific antibodies to hsc70 (anti-hsc70) and Impα2 (anti-Impα2), as indicated. A, CLSM images of cells after 18 min are shown; exclusion of TR70 demonstrates nuclear integrity. B, CLSM images such as those shown in A were analyzed using ImageJ software to determine the nuclear to cytoplasmic ratio (Fn/c). Results represent the mean ± S.E. from a single typical experiment, with each data point representing more than three separate measurements of Fn and Fc above background. Curves were fitted (3 parameter sigmoidal) using SigmaPlot software. Schematic representations of the SRY HMG (high mobility group) domain, the CaM-binding and the Impβ1 binding NLS, with effects of sex-reversing mutations indicated, are shown above. C, pooled data (mean ± S.E., n > 2) for the percentage maximal nuclear accumulation, Fn/c_max, in the presence and absence of indicated antibodies. D, pooled data (mean ± S.E., n > 2) for the initial rate of nuclear accumulation (Fn/c s⁻¹) in the presence and absence of indicate antibodies. p values are indicated where there were significant differences compared with in the absence of the indicated antibodies. No Add., no addition.