Panel A shows a vertical bar graph comparing the climbing ability of flies of different genotypes. The y-axis represents the percentage of flies, and the x-axis lists the genotypes. The w1118 genotype shows a significantly higher percentage of flies climbing past the 5-centimeter mark within 2 seconds compared to the ana1 mutant. Panel B presents a vertical bar graph quantifying the number of centriole pairs marked by Asl localization in cells from different genotypes. The y-axis indicates the number of centriole pairs, and the x-axis lists the genotypes. The data points show no significant difference between the genotypes. Panel C displays Western blots showing the pull-down of EGFP-Ana1 from S2 cells incubated with testis lysates from w1118 or cep135c04199 mutants. The blots are probed with anti-Cep135 and anti-tubulin antibodies. The residual Cep135 in mutants fails to bind Ana1. Panel D shows a schematic representation of various Ana1 and Cep135 constructs analyzed for binding. Panel E presents isothermal titration calorimetry (ITC) graphs showing the interaction between different fragments of Cep135 and Ana1. Each graph shows the binding affinity (Kd) and the interaction over time.
Mapping of the interaction region between Cep135 and Ana1. (A) Climbing ability was assessed by measuring the percentage of flies per genotype that climbed past the 5-cm mark within 2 s. Each data point represents the percentage from 20 flies, with three independent biological replicates using distinct cohorts of flies per genotype (total of 60 flies per genotype). Error bars indicate the mean ± SD. ****P < 0.0001 (unpaired two-tailed Student’s t test). (B) Centriole pairs, marked by Asl localization, were quantified in 15–20 cells per testis from the indicated genotypes. Each data point represents the number of centriole pairs per testis, derived from five independent testes per genotype. Error bars indicate the mean ± SD. ns, not significant (unpaired two-tailed Student’s t test). (C) Pull-down of EGFP-Ana1 from S2 cells incubated with testis lysates (150 testes/genotype) from w1118 or cep135c04199 mutants. Blots were probed with anti-Cep135 (aa1–225). α-Tubulin is a loading control. The residual Cep135 in mutants (Cep135-N*) fails to bind Ana1. (D and E) ITC-based experiments to screen for interactions between Cep135 and Ana1 fragments, and a schematic representation showing various Ana1 and Cep135 constructs analyzed for binding in this study. Source data are available for this figure: SourceData FS1.