Extended Data Fig. 7: The circulating levels of dilp2 and AKH in haemolymph are measured by mass spectrometry and dot blot assay.
From: A glucose-sensing neuron pair regulates insulin and glucagon in Drosophila
a, Sequences of dilp2, AKH, tryptic peptide of dilp2 B chain and tryptic peptide of AKH. We detected dilp2 B chain at m/z (mass to charge ratio): 369.1785 (TLCSEK, M2H+: 369.1785) and AKH at m/z: 497.2374 (QLTFSPDW, M2H+: 497.2374). b, Nomenclature and m/z values of fragment ions (N-terminal directed ‘a’ and ‘b’ ions, as well as, C-terminal directed ‘y’ ions) which is driven by dilp2 B chain and AKH. c, d, Relative extracted ion intensities for the dilp2 B chain and its fragment ions in each trial (c), and AKH and its fragment ions in each trial (d) generated from the haemolymph of fed flies in which CN neurons were inactivated, or those of control flies; see Methods. e, f, A dot blot (e) and its quantification (f) show the levels of dilp2 in the haemolymph of wild type (w1118), UAS-Kir2.1/CN-Gal4;dilp2-HA and UAS-Kir2.1/+;dilp2-HA flies, probed with anti-HA antibody to detect dilp2. Because w1118 flies do not express dilp2–HA, they were used as a negative control. g, h, A dot blot (g) and its quantification (h) show the levels of AKH in the haemolymph of CN-Gal4/+, UAS-Kir2.1/+ and UAS-Kir2.1/CN-Gal4 flies, probed with anti-AKH antibody. The intensity of black dots in the red dashed circle represents the quantity of dilp2 or AKH that was later normalized to Ponceau staining. For gel source data, see Supplementary Fig. 1. **P < 0.01 and ***P < 0.001; unpaired two-tailed t-test (f) and one-way ANOVA with Tukey post hoc test (h). See Supplementary Table 1 for the sample sizes and statistical analyses.
