Fig. 2. NUR is a secreted protein and its ectopic expression induces sleep.

(A) SEAP assay. Left: Culture medium from S2R+ cells expressing alkaline phosphatase (AP), NUR::AP, or a fusion of AP with the immunoglobulin binding protein (BiP), BiP::AP, used as a secreted control, with (+) or without (−) induction by Cu²⁺ in a 96-well plate. The arrow indicates NUR expression in the media. Right: Quantification of the SEAP assay by measuring absorbance at 595 nm for each sample (A.U., absorbance units). ****P < 0.0001 (Student t test). Error bars denote SEM. (B) Protein immunoblot of S2R+ cells (left) or conditioned medium (right) using antibodies to GFP. Cells expressed myr::GFP (negative control), NUR::GFP, or BiP::GFP (positive control); with (+) or without (−) induction by Cu²⁺.The arrow indicates the detection of NUR::GFP in the medium. (C) Sleep profiles and expression patterns of Gal4 lines ectopically expressing nur in various subsets of neurons in the brain. Upper panels show the sleep pattern of each genotype: Gal4/UAS-nur (red), Gal4 control (blue), and UAS-nur control (green). The average number of minutes of total sleep in eight female flies is indicated. Lower panels show the expression pattern of each Gal4 line, as determined by expression of UAS-mCD8::GFP (green) and costaining with nc82 (neuropile marker; magenta) (images from Brainbase: https://braingazer.org/).