(A) A schematic of the discs-large1 (dlg1) gene region (based on FlyBase annotation release FB2014_05). The site of MI06353 insertion is shown with the red triangle. Each isoform is labeled by name and molecular weight of the protein product. Isoforms tagged with EGFP at the MiMIC insertion site have black labels, green boxes (coding exons) and blue boxes (3′-, 5′-UTR exons); while isoforms not tagged with EGFP have red labels with orange and brown exon boxes. The black bars represent each protein domain as they map on the genetic sequence. (B) Western blots of head extracts from control: y w and y w dlg1-EGFP-dlg1 were probed with anti Dlg1 (upper), which recognizes the second PDZ domain, and anti GFP (lower). (C) A diagram representing temperature conditions used in subsequent experiments to modify protein expression levels. The top bar indicates developmental stages: E (embryo), L1 (first instar larva), L2 (second instar larva), L3 (third instar larva), P (pupa), A (adult). The next bar is a time line (in days) of the developmental stages for animals kept at 18°C. The bars below indicate the time at which the animals were shifted to 28°C at the beginning of first, second or third instar, or kept continuously at 28°C. (D) Third instar larval brains stained with Dlg1 (a and d) and GFP (b and e) antibodies. y w dlg1-EGFP-dlg1;;UAS-NSlmbvhhGFP4/tub-GAL4 animals that were raised continuously at 18°C show robust larval brain expression of Dlg1 (a–c) with complete colocalization of Dlg1 and GFP expression (c). However, animals that were shifted from 18°C to 28°C as second instar larvae (see C) have less Dlg1 expression as judged by both Dlg1 (d) and GFP (e) antibody staining, however colocalization of Dlg1 and GFP is still present in some areas (f). Additionally, these brains (d–f) are significantly larger compared with controls (a–c), which is a characteristic phenotype associated with loss of function alleles of dlg1. The white arrows point to neuromuscular junctions. Scale bar, 100 μm. (E) Wing discs from third instar larvae labeled with GFP and E-cad antibodies. First instar larvae were shifted from 18°C to 28°C. The larvae that ubiquitously express deGradFP, y w dlg1-EGFP-dlg1;;UAS-NSlmbvhhGFP4/tub-GAL4 (b) have significantly larger wing discs compared with controls minimally or not expressing deGradFP, y w dlg1-EGFP-dlg1;;UAS-NSlmbvhhGFP4/+ (a), another traditional dlg1 mutant phenotype. Scale bar, 100 μm.