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1.
FIGURE 6.

FIGURE 6. From: 70-kDa Heat Shock Cognate Protein hsc70 Mediates Calmodulin-dependent Nuclear Import of the Sex-determining Factor SRY.

Retention of SRY in the nucleus is independent of hsc70. A and B, representative CLSM images of recovery of nuclear fluorescence after photobleaching in HeLa cells transiently transfected to express full-length GFP-SRY-wild type (WT; A) and GFP-SRY-R133W (B) treated with or without siRNA to hsc70 over time (s), after photobleaching (post-bleach). The yellow dotted line indicates the bleached area (∼50% of nucleus). C, quantification of the recovery over time of nuclear fluorescence after photobleaching (Fnbleach, expressed in terms of fractional recovery of respective time points relative to the Fnnon-bleach prebleach value). Results shown are for a representative cell. D, pooled data for the mean ± S.E. (n = 3) percentage maximal recovery of Fnbleach, normalized to 0 s post-bleach.

Gurpreet Kaur, et al. J Biol Chem. 2013 February 8;288(6):4148-4157.
2.
FIGURE 2.

FIGURE 2. From: 70-kDa Heat Shock Cognate Protein hsc70 Mediates Calmodulin-dependent Nuclear Import of the Sex-determining Factor SRY.

SRY nuclear import is enhanced by hsc70 and CaM. Nuclear import of GFP-SRY-HMG-wild type (WT) and its mutant derivatives was reconstituted in vitro as per the legend to Fig. 1, with or without (No Add.) 1.5 μm hsc70 and/or CaM. CLSM images of cells at 18–20 min are shown (top panels), with TR70 indicating intact nuclei, for GFP-SRY-HMG-WT (A), the CaM-NLS (B), and β-NLS (C) mutant derivatives, with quantitative analysis of such images (bottom panels) as per the legend to Fig. 1. D and E, pooled data (mean ± S.E., n = 3) for percentage maximal nuclear accumulation (Fn/cmax) (D) and initial rate of nuclear accumulation (E) determined as per the legend to Fig. 1, in the presence and absence of the various additions for the indicated GFP-SRY-HMG derivatives. p values are shown where there were significant differences compared with in the absence of the addition of CaM and hsc70 proteins.

Gurpreet Kaur, et al. J Biol Chem. 2013 February 8;288(6):4148-4157.
3.
FIGURE 5.

FIGURE 5. From: 70-kDa Heat Shock Cognate Protein hsc70 Mediates Calmodulin-dependent Nuclear Import of the Sex-determining Factor SRY.

Efficient nuclear import of SRY is dependent on hsc70 expression. A, representative CLSM images of the return of nuclear fluorescence after photobleaching in HeLa cells transfected to express full-length GFP-SRY- wild type (WT) in the presence or absence of siRNA to hsc70 over time (s) after photobleaching (post-bleach). Yellow dotted lines indicate regions of the nucleus photobleached. B, images such as those in A were quantified for the recovery over time of the nuclear to cytoplasmic fluorescence ratio (Fn/c) after photobleaching, expressed in terms of fractional recovery of respective time points divided by the initial (prebleach) value. Results shown are for a representative cell. C, pooled data for the mean ± S.E. (n > 13) for the percentage maximal recovery (left panel; normalized to 0 s post-bleach), time (s) taken to reach half maximal recovery (t½) (middle panel) and initial rate of recovery (right panel) in the presence and absence of siRNA to hsc70 for GFP-SRY-WT and GFP-T-ag NLS. p values are indicated where the values are significantly different in the presence of hsc70 siRNA.

Gurpreet Kaur, et al. J Biol Chem. 2013 February 8;288(6):4148-4157.
4.
FIGURE 3.

FIGURE 3. From: 70-kDa Heat Shock Cognate Protein hsc70 Mediates Calmodulin-dependent Nuclear Import of the Sex-determining Factor SRY.

hsc70 specifically binds the SRY/CaM complex in Ca2+-dependent fashion. A, native PAGE gel mobility shift assay (see “Experimental Procedures”) for hsc70 and CaM binding to GFP-SRY-HMG (wild type) was performed, where GFP-SRY-HMG (1 μm) was incubated in the absence and presence of hsc70 (1 μm) and/or CaM (1 μm) in the presence of Ca2+ (2 mm) for 20 min at room temperature as indicated, and then fluorimaging was performed subsequent to gel electrophoresis. Results are shown for a single typical assay from a series of three similar experiments, with SRY·CaM and SRY·CaM·hsc70 complex formation indicated on the right. B, immunoprecipitation using GFP-TRAPTM resin was performed as described under “Experimental Procedures,” with immunoblots probed with anti-GFP or anti-hsc70 antibodies to detect binding of GFP or GFP-SRY-HMG wild type and NLS mutant derivatives in the absence and presence of 2 μm Ca2+ as indicated to hsc70. I, input. C, densitometric analysis for immunoprecipitation assays was performed on images such as those in B for hsc70 binding to the GFP-SRY-HMG and NLS mutant derivatives in the presence of Ca2+, as indicated. Results represent the mean ± S.E. (n = 3) of the percentage hsc70 bound relative to GFP-SRY in the presence of Ca2+, with p values shown where there were significant differences compared with wild type.

Gurpreet Kaur, et al. J Biol Chem. 2013 February 8;288(6):4148-4157.
5.
FIGURE 4.

FIGURE 4. From: 70-kDa Heat Shock Cognate Protein hsc70 Mediates Calmodulin-dependent Nuclear Import of the Sex-determining Factor SRY.

siRNA against hsc70 inhibits CaM-dependent nuclear import of SRY. A, immunoblot analysis (left panel) for the protein levels of hsc70 of HeLa cells treated with hsc70-specific small interfering RNA duplexes (hsc70 siRNA) 72 h post-treatment and probed with anti-hsc70 and anti-α/β tubulin antibody. Densitometric analysis (right panel) of images such as those in the left panel was performed to quantify the relative amount of hsc70 protein in the absence and presence of siRNA to hsc70 standardized relative to α/β-tubulin in whole cell extracts. Results are for a single typical experiment from a series of three similar experiments. B, HeLa cells were transiently transfected to express the indicated GFP-fusion proteins, 48 h post-treatment with siRNA to hsc70, and imaged live 20 h post-transfection by CLSM. C, results for the quantitative analysis, whereby CLSM images such as those in A were analyzed using ImageJ software for the nuclear to cytoplasmic ratio (Fn/c; fold extent of nuclear accumulation). Results are for the mean ± S.E. (n > 44) of a single typical experiment from a series of three similar experiments; p values are indicated where the Fn/c values are significantly different in the presence of hsc70 siRNA. WB, Western blot.

Gurpreet Kaur, et al. J Biol Chem. 2013 February 8;288(6):4148-4157.
6.
FIGURE 1.

FIGURE 1. From: 70-kDa Heat Shock Cognate Protein hsc70 Mediates Calmodulin-dependent Nuclear Import of the Sex-determining Factor SRY.

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/cmax, 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.

Gurpreet Kaur, et al. J Biol Chem. 2013 February 8;288(6):4148-4157.

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