Panel A shows SDS-PAGE analysis for total, input, and aggregate fractions from ovaries treated with different concentrations of azetidine-2-carboxylic acid. The gel displays protein bands at various molecular weights, with notable bands around 50 and 37 kilodaltons. Panel B is a bar graph depicting the ratios of aggregated fractions relative to total protein at different azetidine-2-carboxylic acid concentrations. The x-axis represents ACA concentration in millimolar (0 mM, 5 mM, 10 mM), and the y-axis represents the ratio [agg]/[total]. The graph shows an increase in the ratio with higher ACA concentrations, with a significant difference indicated by an asterisk. Panel C shows SDS-PAGE analysis for the gel displays protein bands at various molecular weights, with notable bands around 50 and 37 kilodaltons. Panel D is a bar graph depicting the ratios of aggregated fractions relative to total protein at different canavanine concentrations. The x-axis represents canavanine concentration in millimolar (0 mM, 0.1 mM, 0.5 mM, 1.0 mM), and the y-axis represents the ratio [agg]/[total]. The graph shows an increase in the ratio with higher canavanine concentrations, with a significant difference indicated by an asterisk. Panel E shows Western blot analysis for H3 levels at different fractions of the aggregate isolation assay in WT and NASP stage 14 egg chambers. The blot displays protein bands at various molecular weights, with notable bands around 20, 15, 37, and 25 kilodaltons.
Validation of aggregate isolation assay. (A) SDS-PAGE analysis for total, input, and aggregate fractions from ovaries collected from WT female flies and treated with different concentrations of azetidine-2-carboxylic acid. (B) Ratios of the aggregated fractions relative to the total protein at different azetidine-2-carboxylic acid concentrations quantified from the SDS-PAGE. N = 3 biological replicates. Data represent the mean ± standard deviation (SD). Statistical difference between groups was analyzed by employing a one-way ANOVA Dunnett’s multiple comparisons test. (C) SDS-PAGE analysis for total, input, and aggregate fractions from S2 cells treated with different concentrations of canavanine. (D) Ratios of the aggregated fractions relative to the total protein at different canavanine concentrations quantified from the SDS PAGE. N = 4 biological replicates. Data represent the mean ± standard deviation (SD). Statistical difference between groups was analyzed by employing a one-way ANOVA Dunnett’s multiple comparisons test. (E) Western blot for H3 levels at different fractions of the aggregate isolation assay in WT and NASP stage 14 egg chambers. The total and cell debris fractions are diluted 1:10 to prevent saturation of the chemiluminescent signal. In the figure panels, ns represents a nonsignificant difference, while *P < 0.05. Source data are available for this figure: SourceData FS4.