Identification and Functional Characterization of N-Terminally Acetylated Proteins in Drosophila melanogaster
Figure 5
In vivo analysis of genetically modified hyrax transgenes.
For an in vivo analysis, the hyrax Hyx-A2P-HA, Hyx-Wt-HA transgenes were integrated into the fly genome using the site-specific phiC31-mediated integration system [36]. Fly genotypes: tub>hyx-wt-HA/CyO; hyx2/TM6b (lanes labeled WT), tub>hyx-A2P-HA/CyO; hyx2/TM6b (lanes labeled A2P), Sp/CyO; hyx2/TM6b (negative controls, lanes labeled hyx). Numbers in lanes represent µg of protein loaded (A, B). (A) Total fly lysates of flies reared at 25°C were subjected to Western blot analysis and revealed identical expression levels for the two transgenes (97% Hyx-Wt-HA in comparison to Hyx-A2P-HA). Western blot analysis of total fly lysates of flies reared at 29°C revealed a 32% reduced amount of Hyx-A2P-HA protein as compared to Hyx-Wt-HA controls. (B) Confocal images of stained wing discs from 3rd instar larvae of Sp/CyO; hyx2/TM6b (negative controls, opened pinhole), Hyx-Wt-HA/CyO; hyx2/TM6b and Hyx-A2P-HA/CyO; hyx2/TM6b reared at 25°C and 29°C, respectively. At both temperatures, a similar strong nuclear staining was observed for Hyx-Wt-HA and Hyx-A2P-HA and no difference in localization could be detected.