Fig. 4: Vps15 interacts with Bmal1.
From: Class 3 PI3K coactivates the circadian clock to promote rhythmic de novo purine synthesis
a, Immunoblots of Bmal1-containing immunoprecipitates from total extracts of MEFs. Right: clock levels normalized to Bmal1 in immunoprecipitates presented as the mean ± s.e.m. (independent repeats, n = 3). b, ChIP–qPCR analysis of Bmal1 recruitment to the indicated gene promoters in GFP and CRE MEFs 24 h post dexamethasone synchronization. Data are the mean ± s.e.m. fold enrichment from three independent experiments (n = 6 for GFP-IgG, n = 7 for GFP-Bmal1 and n = 8 for all other groups). c, Left: immunoblot of BMAL1 immunoprecipitates from the total extracts of HEK293T cells transfected with increasing amounts of VPS15WT. Right: CLOCK levels normalized to BMAL1 in BMAL1 immunoprecipitates are presented as the fold difference to the cells transfected with empty vector. Data are the mean (independent repeats, n = 2 for VPS15WT–Flag 2.5 μg and n = 3 for all other groups). d, Immunoblot of VPS15–Flag-containing complexes immunoprecipitated from HEK293T cells transfected with the indicated constructs. e, Proximity ligation assay of endogenous VPS15 and Flag–BMAL1 in HEK293T cells (co-transfected with GFP). The ‘no antibodies’ condition served as the negative control. Scale bar, 10 µm. Right: data are the mean ± s.e.m. number of proximity puncta per cell (n = 8 no antibody and n = 25 anti-Flag + anti-VPS15 fields) from three independent experiments (>500 cells per condition). b,e, *P < 0.05; two-tailed unpaired Student’s t-test. f, Immunoblot, using the indicated antibodies, of Vps15 immunoprecipitates from the soluble nuclear fractions of liver tissue of WT 5-week-old male mice (ZT6; fed ad libitum). g, Immunoblot analyses of VPS15WT- and VPS15Mut1-interacting proteins from HEK293T cells immunoprecipitated with anti-Flag. h, Left: immunoblot of the total extracts of GFP and CRE MEFs transduced with adenoviral vectors expressing GFP, VPS15WT or VPS15Mut1 and synchronized by dexamethasone. Right: densitometric analyses of Rev-Erbα normalized to Actin presented as the fold change compared with the GFP-GFP condition. Data are the mean ± s.e.m. from three independent repeats. *P < 0.05 for GFP versus CRE-GFP and **P < 0.05 for CRE-VPS15WT/Mut1 versus GFP-CRE; two-tailed unpaired Student’s t-test. i, Left: ___domain organization of mouse full-length Bmal1 protein (Protein Data Bank, 4F3L). Right: mapping of its truncated constructs. Middle: Immunoblot of Bmal1 immunoprecipitates from total extracts of HEK293T cells. The experiment was performed three times. Representative blots are shown. aa, amino acids. Source data and unprocessed blots are provided.
