Figure 4.

BDBT does not exhibit detectable daily oscillations in level in the eyes of wild-type flies and is not notably lower in the eyes of arr mutants than in wild-type eyes

Heads were isolated at the indicated times of day from wild-type Canton S and arr1¹ or arr2³ mutants, and extracts of the heads were assayed by immunoblot analysis for PER and BDBT (Figure S2). Here, heads were dehydrated in acetone and dissected after drying for analysis of BDBT, DBT, and tubulin levels by immunoblot analysis. One representative analysis is shown in panel A. The molecular weight of markers is indicated to the left. 4–5 separate analyses were analyzed with scans of the chemiluminescent signals. Each BDBT signal was normalized to the signal for tubulin from the same gel lane, and this normalized signal was further normalized to the tubulin-normalized signal for Canton S wild-type flies at ZT19 (which therefore had a signal of 1 for each analysis). The averages are plotted here with the standard error of the mean shown as a bracket above the bar. Statistical analysis (ANOVA(11,42) = 1.59, p = 138.16) with a Tukey HSD test showed no statistically significant differences between normalized BDBT levels in any of the samples, and likewise statistical analysis of each genotype time course alone showed no statistically significant differences. The variations in BDBT immunofluorescence seen over the course of the day in wild-type flies are suggested to be caused by antigen-masking effects because high levels of BDBT immunofluorescence are detected at ZT7 in wild-type flies when the sections are subjected to 60°C temperatures prior to antibody application (Figure S3).