Extended Data Fig. 5: Rhythmic nuclear expression of class 3 PI3K subunits.
From: Class 3 PI3K coactivates the circadian clock to promote rhythmic de novo purine synthesis
a, Immunoblot analysis, using the indicated antibodies, of the soluble nuclear and cytosolic fractions of HEK293T cells. GAPDH and LAMIN A/C served as purity controls for the cytosolic and soluble nuclear fractions. Experiment repeated five times; representative repeat shown. b,c, Immunoblot analysis of soluble nuclear (b) and euchromatin (c) fractions from the livers of eight-week-old male WT mice. Densitometric analysis of Vps15 and Vps34 normalized to total protein (TGX gels) are presented as the fold change over ZT0. Data are the mean ± s.e.m. (n = 4 mice). Rhythmicity pattern was tested using JTK_CYCLE (Supplementary Table 1). d, Mean normalized Vps15 and Vps34 protein levels in soluble nuclear (as in b) and euchromatin (as in c) fractions during the light (ZT0–ZT8) and dark (ZT12–ZT20) phase. Data are the mean ± s.e.m. (n = 8 mice in dark and n = 16 mice in light). #P < 0.05 versus light; two-tailed unpaired Student’s t-test. e, Relative transcript levels of the indicated genes in the livers of mice treated as in b. Data are presented as the mean ± s.e.m. (n = 4 mice). Rhythmicity was determined using JTK_CYCLE (Supplementary Table 1). f, Immunoblot analysis, using the indicated antibodies, of the soluble nuclear protein extracts of MEF cells treated with ivermectin for the indicated times. Densitometric analyses of Vps15 and Vps34 normalized to lamin B1 presented as the fold change over vehicle-treated cells. Data are the mean ± s.e.m. (independent repeats: n = 7 ivermectin (4 h) and n = 8 for all other groups). #P < 0.05 versus DMSO; two-tailed unpaired Student’s t-test. g, Immunoblot analyses of IPOA5 co-immunoprecipitated with ectopically expressed VPS15 and VPS34 from HEK293T cells using anti-Flag (ev, empty vector). Immunoprecipitation was performed three times; representative blots shown. h, Proximity ligation assay between endogenous VPS15 and ectopic Flag–VPS34 protein in HEK293T cells (co-transfection with GFP-expressing vector visualized transfected cells). The ‘no antibody’ condition served as a control for the non-specific signal. Scale bar, 10 µm. Data are the mean ± s.e.m. of proximity puncta per cell (n = 8 fields (no antibody) and n = 14 fields (Flag + VPS15) with over 300 cells collected in three independent experiments for each condition is presented; no antibody condition shared with Fig. 4e). #P = 0.00005 versus no antibody; two-tailed unpaired Student’s t-test. Source numerical data and unprocessed blots are provided.
