Figure 1.
Relocalization of nuclear pseudophosphorylated αB-crystallin in response to heat-shock.
Transiently transfected HeLa cells expressing αB-STD were mock-treated or subjected to heat-shock and allowed to recover for 6 or 24 hours. The localization of αB-STD was visualized by immunofluorescence, using a monoclonal antibody against αB-crystallin.
Figure 2.
Pseudophosphorylated αB-crystallin enhances refolding of heat-inactivated nuclear luciferase.
Transiently transfected HeLa cells expressing nuclear luciferase were heated (45 min at 45oC) and subsequently lysed directly or 6 hours thereafter. Cycloheximide was added just prior to heating to prevent de novo luciferase synthesis. Recovery of luciferase activity is indicated for cells cotransfected with empty pIRES vector (pIRES), or constructs encoding wild-type (WT), non-phosphorylatable αB-crystallin (αB-STA) or pseudophosphorylated αB-crystallin (αB-STD). The luciferase activity was determined and compared with the initial luciferase activity before heat shock. All transfections were corrected for the transfection efficiency as measured by the β-galactosidase activity resulting from a cotransfected β-galactosidase expression vector. The results represent the mean values of 6 independent experiments; error bars indicate the standard deviation. A western blot illustrating the expression of αB-crystallin is shown in Figure S1.
Figure 3.
Co-immunoprecipitation of Gemin3 with αB-crystallin.
Extracts of HeLa cells, transfected with pIRES as a control or pIRES constructs coding for wild-type (WT), non-phosphorylatable αB-crystallin (αB-STA) or pseudophosphorylated αB-crystallin (αB-STD) were subjected to immunoprecipitation with a monoclonal antibody against αB-crystallin. The immunoprecipitates were analyzed by immunoblotting using a mouse monoclonal antibody against Gemin3 and rabbit polyclonal antibodies to αB-crystallin. Note that Gemin3 shows two bands, of which the lower band is likely a degradation product.
Figure 4.
The effect of knockdown of Gemin3 and Gemin2 on the nuclear import of pseudophosphorylated αB-STD.
Doxycycline-inducible T-REx™-HeLa-αB-STD cells were treated with two different Gemin3 siRNAs, two different Gemin2 siRNAs and two negative control siRNAs (LUC and POP1). After 24 hours 1 µg/µl doxycycline was added to induce αB-STD expression and 16 hours later the cells were analyzed. (A) Knockdown of Gemin3 and Gemin2 was assessed by western blotting analysis. αB-crystallin and γ-tubulin were used as loading controls. (B) T-REx™-HeLa-αB-STD cells treated with the different siRNAs were stained for αB-crystallin. (C) The percentages of the siRNA-treated cells containing αB-crystallin-positive speckles in the nucleus were determined and are shown in the graph. Statistical analysis was performed using One-way ANOVA and Tukey’s Multiple Comparison Test. *** P<0.001, ** 0.001<P<0.01, * 0.01<P<0.05.
Figure 5.
In vitro nuclear import of pseudophosphorylated αB-crystallin is dependent on the SMN complex.
In vitro nuclear import assays were conducted by incubating digitonin-permeabilized HeLa cells for 1 hour at room temperature with reticulocyte lysates containing ATP and GTP. (A) Import reactions were performed with FITC-labeled recombinant wild-type αB-crystallin (WT), non-phosphorylatable αB-crystallin (αB-STA) and pseudophosphorylated αB-crystallin (αB-STD). As a negative control the import assay with αB-STD was conducted at 4°C (STD 4°C). (B) Import reactions of αB-STD with rabbit reticulocyte lysates immunodepleted with anti-Gemin3 antibodies (ΔGemin3), anti-SMN antibodies (ΔSMN) and control-depleted (Cntr). The impaired import of αB-STD with SMN-depleted reticulocyte lysate could be rescued by addition of purified SMN complex (ΔSMN+ SMN complex). (C) The immunodepleted reticulocyte lysates were analyzed by western blot to assess depletion. Skp1 was used as a loading control. Dotted circles indicate the position of the nuclei.