GFP::HDEL distribution. (A) Representative examples of confocal projections of WT larvae showing the distribution of the ER markers tdTom::Sec61β and GFP::HDEL in a Type Ib motor neuron. Genotype: Rtnl1⁺/UAS-tdTom::Sec61β; Ib-GAL4/UAS-GFP::HDEL. (B) Quantification of the ER marker GFP::HDEL in Type I NMJs (muscles 1/9 NMJ) of WT, Het, Rtnl1¹⁸, and T-Het larvae. Genotypes: WT—Rtnl1⁺; Ib-GAL4, UAS-CD4::tdTom/UAS-GFP::HDEL Het - Rtnl1⁺/Rtnl1¹⁸; Ib-GAL4, UAS-CD4::tdTom/UAS-GFP::HDEL Rtnl1¹⁸ - Rtnl1¹⁸; Ib-GAL4, UAS-CD4::tdTom/UAS-GFP::HDEL T-Het - Rtnl1¹⁸/Rtnl1¹ ; Ib-GAL4, UAS-CD4::tdTom/UAS-GFP::HDEL. Plots show individual larval datapoints and mean ± SEM; y-axis indicates arbitrary units (au) after normalization to control (WT); intensity values are relative to CD4::tdTom signal; sample size (number of larvae) is indicated within the plots for each genotype. For each larva, several NMJs between A2-A6 segments were analyzed, and the mean value is shown as a larval datapoint. (C) Representative examples showing the selection of GFP::HDEL puncta in confocal projections of Type Ib muscle 1 NMJ (magnified areas from Fig. 3 C). HiLo lookup table (LUT) shows saturated pixels in red and pixels with no detectable signal in blue. Intermodes thresholding of GFP::HDEL intensity was used to select only those regions with high GFP::HDEL levels (region of interest, ROI). Genotypes are Ib-GAL4, UAS-CD4::tdTom/UAS-BiP::sfGFP::HDEL, in either a Rtnl1⁺ (WT) or Rtnl1¹⁸ background.