Quantification of pAkt levels

Quantification of pAkt immunoreactivity was performed on each L3 wing disc. In all cases, six or more wing discs were dissected and imaged for each genotype. Images were acquired with a Zeiss LSM 880 with a 34-channel spectral Quasar detector. Representative images of wing discs were processed in Adobe Photoshop CS5 (Adobe Systems, San Jose, CA). To quantify pAkt levels, all tissues were processed with the same reagents, and then imaged and analyzed in identical ways. For each image, 3 z-stacks were selected immediately under the peripodial cell layer from the apical region of each wing disc, to create a maximum projection image that was quantified using Image J. The pAkt intensity levels were reported as a posterior/anterior intensity ratio, where an identical square was drawn within each posterior and anterior compartment of the wing disc to measure the intensity in the same region. Statistical analysis and graphs of datasets were performed using GraphPad Prism 8 (GraphPad Software, San Diego, CA) and data within graphs were compared using one-way ANOVA followed by Tukey post hoc analysis. Differences between groups were considered statistically significant when p<0.05, since ANOVA corrects for multiple testing. Data are presented as mean ± Standard Deviation (SD).