Fig. 4: vaha mutants are defective in ILP2HF secretion.

a Immunostaining of brains from 5-7 day old control, vaha mutant and rescue flies for ILP2HF (anti HA, red) and DAPI (blue). The genotypes are control w^-; +/+; gd2HF/+; vaha mutant w^-; vaha/vaha; gd2HF/+ and rescue w^-; NP1 Gal4.vaha/vaha; gd2HF/UAS VahaGFP. Flies fasted overnight are fed either regular food (Reg) or regular food containing coconut oil (HFD) for 1 h. Confocal images are projection of z stacks n = 6 brains, scale bar 50 μm. b Quantification of ILP2HF fluorescence intensity in the IPCs of control, vaha mutants and rescue flies in the fasted and fed states (described in a). Each data point represents fluorescent intensity in one IPC, n = 47 (control fasted), n = 51(control fed Reg), n = 48 (control fed HFD), n = 47 (mutant fasted), n = 50(mutant fed Reg), n = 52(mutant fed HFD), n = 49 (rescue Reg) and n = 49 (rescue HFD). One-way ANOVA followed by Tukey’s multiple comparison test is used and horizontal line indicates median. c ILP2HF levels in circulation assayed in control, vaha mutants and rescue in the fasted and fed states (genotypes described in a). Each data point represents value from n = 3, 15 male flies per replicate. One-way ANOVA followed by Tukey’s multiple comparison test is used and data presented as mean ± SD. d Glucose content e TAG content measured in homogenates from flies described in a. Each data point represents value from 10 flies. n = 3, data is assessed by two-tailed Student’s t-test and presented as mean ± SD. Rescue is shown for flies on regular food. f IPC activity measured in control and vaha mutants using the CaLexA system. The genotype of control flies is w^-; LexAopCD8GFP2ACD8GFP/dILP2Gal4; CaLexALexAop CD2GFP/gd2HF and mutant flies is w^-; LexAopCD8GFP2ACD8GFP.vaha /dILP2Gal4.vaha; CaLexALexAopCD2GFP/gd2HF. Confocal images are projection of z stacks, n = 6 brains, scale bar 20 μm. g Each dot represents intensity of GFP fluorescence in one brain, flies are described in f. n = 6 brains. Data is assessed by One-way ANOVA followed by Tukey’s multiple comparison test and horizontal line indicates median. Source data are provided for 4b, c, d, e, g as a Source data file.