Quantitation of row 1 proteins during after tubocurarine block in culture

Each file is for a single mature row 1 complex member, examining its distribution in mouse inner hair cells in the presence or absence of transduction channel block. Cochleas were dissected at P4.5 and cultured in in vitro for two days in control solution or 100 µM tubocurarine. The first two to three tabs in each file represent data from different experiments. Each block of 12 measurements is from a single hair bundle. The next-to-last tab has the average of all row 1 + 2 measurements, giving total expression in those rows. The last tab compares rows 1 and 2.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 control and tubocurarine block, the average signal for control bundles on a given experimental day was used to normalize all samples, allowing comparison of controls and channel block with identical acquisition parameters.