Quantitation of redistribution of row 1 and 2 proteins in transduction mutants

Krey et al. Fig. 5 data.zip contains spreadsheets with fluorescence quantitation for row 1 proteins in tips of mice from transduction mutants.
Krey et al. Fig. 8 data.zip contains spreadsheets with fluorescence quantitation for row 2 proteins in tips of mice from transduction mutants.
Krey et al. Fig. S4 data.zip contains spreadsheets with fluorescence quantitation for row 1 proteins in tips of mice from aider range of genotypes of transduction mutants.
We estimated the total immunofluorescence signal in the top 1-2 µm of each tip. Airyscan z-stacks were imported into Fiji Software, which was used for all analysis steps. For analysis of P7.5 mutant mice, average Z-projections of Airyscan stacks were made that included row 1 and row 2 tips in the same projection. For analysis of P21.5 mutant mice, due to increased height variability, Regions of Interest (ROIs) were selected at row 1 or row 2 tips within individual x-y slices from the z-stacks. Images were kept as multi-channel stacks and phalloidin was used to guide selection at the tips of each row. Regions of interest (ROIs) used circles that encompassed most of each tip. Area and average intensity measurements were made from ten or more row 1 tips and ten or more row 2 tips, and blank measurements were taken from volumes outside the stereocilia and above the epithelium. The total signal (area times average intensity) in each stereocilia tip volume was calculated after subtracting the background. For each hair bundle, the average signal in rows 1 and 2 was calculated, which was used to normalize the individual row 1 and row 2 stereocilia tip signals within each bundle. The average signal row 1 and 2 signal for each bundle was also used for comparison of expression levels at different developmental times or between control and mutant mice. For comparisons between transduction-mutant genotypes, the average signal for control bundles on a given experimental day was used to normalize all genotypes, allowing comparison of controls and mutants with identical acquisition parameters. .