Figure 5. Panel A: A schematic diagram... | Rockefeller University Press

Figure 5.

$NASP functions in the cytoplasm. (A) Schematic of the aggregate isolation protocol used to separate aggregates from stage 14 egg chambers and early embryos. (B) SDS PAGE analysis for total and aggregated protein fractions in WT ovaries when treated with different concentrations of canavanine. (C) Ratio of protein in the aggregate fraction relative to the total protein in WT ovaries at different canavanine concentrations. N > 4 biological replicates. Data represent the mean ± standard deviation (SD). Statistical difference between the groups was analyzed by employing a one-way ANOVA Dunnett’s multiple comparison test. (D) Western Blot analysis for input and aggregate fractions from stage 14 egg chambers from WT and NASP-mutant female flies. (E) Ratios of H3 and H2B in the aggregate fraction relative to input fractions in stage 14 egg chambers of WT and NASP-mutant female flies. N = 4 biological replicates. Data represent the mean ± standard deviation (SD). Statistical difference between the groups was analyzed by employing a paired t test. (F) Western blot analysis for input and aggregate fractions derived from activated eggs collected from WT or NASP-mutant females crossed to twine-mutant males. (G) Ratios of H3 and H2B in the aggregate fraction relative to input fractions from activated eggs collected from WT or NASP-mutant females crossed to twine-mutant males. N = 4 biological replicates. Data represent the mean ± standard deviation (SD). Statistical difference between the groups was analyzed by employing a paired t test. In the figure panels, ns represents nonsignificant difference, while *P < 0.05, ***P < 0.001 and ****P < 0.0001. Source data are available for this figure: SourceData F5. Refer to the image caption for details.$ Panel A: A schematic diagram illustrates the protocol for isolating protein aggregates from stage 14 egg chambers and early embryos. The process includes buffer addition, homogenization, centrifugation, and ultra-centrifugation steps to separate aggregated proteins. Panel B: SDS-PAGE analysis shows total and aggregated protein fractions in wild-type (WT) ovaries treated with different concentrations of Canavanine (0 mM, 0.1 mM, 1.0 mM, and 5.0 mM). The gel images display protein bands at various molecular weights (kD) for total, input, and aggregate fractions. Panel C: A bar graph presents the ratio of protein in the aggregate fraction relative to the total protein in WT ovaries at different Canavanine concentrations. The y-axis represents the aggregate/total protein ratio, and the x-axis shows Canavanine concentrations. Panel D: Western blot analysis show bands for histones H3 and H2B, with total protein as a control. Panel E: A bar graph displays the ratios of H3 and H2B in the aggregate fraction. The y-axes represent the aggregate/input ratios, and the x-axes show WT and NASP mutant groups. Panel F: Western blot analysis for input and aggregate fractions from activated eggs collected from WT or NASP-mutant females crossed to twine mutant males. The blots show bands for histones H3 and H2B, with total protein as a control. Panel G: A bar graph shows the ratios of H3 and H2B in the aggregate fraction. The y-axes represent the aggregate/input ratios, and the x-axes show WT and NASP mutant groups.

NASP functions in the cytoplasm. (A) Schematic of the aggregate isolation protocol used to separate aggregates from stage 14 egg chambers and early embryos. (B) SDS PAGE analysis for total and aggregated protein fractions in WT ovaries when treated with different concentrations of canavanine. (C) Ratio of protein in the aggregate fraction relative to the total protein in WT ovaries at different canavanine concentrations. N > 4 biological replicates. Data represent the mean ± standard deviation (SD). Statistical difference between the groups was analyzed by employing a one-way ANOVA Dunnett’s multiple comparison test. (D) Western Blot analysis for input and aggregate fractions from stage 14 egg chambers from WT and NASP-mutant female flies. (E) Ratios of H3 and H2B in the aggregate fraction relative to input fractions in stage 14 egg chambers of WT and NASP-mutant female flies. N = 4 biological replicates. Data represent the mean ± standard deviation (SD). Statistical difference between the groups was analyzed by employing a paired t test. (F) Western blot analysis for input and aggregate fractions derived from activated eggs collected from WT or NASP-mutant females crossed to twine-mutant males. (G) Ratios of H3 and H2B in the aggregate fraction relative to input fractions from activated eggs collected from WT or NASP-mutant females crossed to twine-mutant males. N = 4 biological replicates. Data represent the mean ± standard deviation (SD). Statistical difference between the groups was analyzed by employing a paired t test. In the figure panels, ns represents nonsignificant difference, while *P < 0.05, ***P < 0.001 and ****P < 0.0001. Source data are available for this figure: SourceData F5.