Figure 6. Wild-type rhodopsin quantification and localization in heterozygous ninaE mutants expressing rhodopsin with a 1D4 monoclonal antibody tag (Rh1-ID4). A: Lysates equivalent of one head were loaded onto each lane and probed with anti-Rh1–1D4 antibody that specifically recognizes wild-type protein. Arrestin2 was used as the loading control. B: The blot probed with anti-Rh1–1D4 antibody was quantified to show levels of rhodopsin in mutants relative to the wild type. The reduction in levels of soluble wild-type rhodopsin in heterozygous mutant flies is significant compared to controls (p<0.0001). C-E: Dark-reared fly retinas were isolated and stained for F-actin and Rh1–1D4. Wild-type photoreceptors show proper rhodopsin localization to the base of the rhabdomeres. F-H: Flies expressing non-1D4-tagged rhodopsin (+/+) show no staining for rhodopsin, highlighting the specificity of the antibody. I-K: Flies haploid for mutant ninaE^R11 and wild-type Rh1–1D4 show partial proper rhodopsin localization with significant rhodopsin-positive puncta mislocalized to the cell body. Error bars are represented as average % rhodopsin±SD. Each column represents the average of three biologic replicates. Scale bar represents 5 μm.