Extended Data Fig. 9: SLEEPY causes constitutively high sleep need by preferentially associating with and phosphorylating SNIPPs.
From: Quantitative phosphoproteomic analysis of the molecular substrates of sleep need
a, Experimental design for comparing the interactomes of SIK3 and SLEEPY from whole brain lysates. b, Summary of SIK3 and SLEEPY interacting proteins (ip) and preferential interacting proteins (pip). c, Gene-annotation enrichment analysis of 289 SLEEPY-preferential interacting proteins (SLEEPY-pip). GO cellular component enrichment analysis using all 22,262 genes of Mus musculus as reference (Ref). Fisher’s exact with FDR multiple test correction was used to determine statistical significance. Top 10 GO terms of fold enrichment (FDR <0.0001), the gene number of SLEEPY-pip and Ref in each term are shown. d, e, Volcano plots showing phosphorylation changes of all putative AMPK substrates in the Sleepy/wild-type group (d) or from the 28 SLEEPY-pip SNIPPs (e). Orange dotted lines, Q = 0.2. f, In vitro kinase assay of recombinant SLEEPY and SIK3, and immunoblotting with AMPK phosphorylation motif antibody (two independent experiments). g–i, Volcano plot showing comparative analysis of whole brain phosphoproteomes (g), all putative AMPK substrates (h) or from 28 SLEEPY-pip SNIPPs (i) in the HG/vehicle (Slp) group. Orange dotted lines, Q = 0.2. j, Quantitative ∆Ps analysis of 190 hyperphosphorylated proteins and 52 hypophosphorylated proteins in the HG/vehicle (Slp) group. Dotted lines, ∆Ps = ±2.4. k–m, Analysis of absolute EEG power spectra (k), relative EEG power spectra (l) and duration (m) of NREMS, REMS and wake states of Sik3Slp/+ (Slp, n = 14) mice injected with vehicle (Veh) or 8 mg/kg HG at ZT6 and ZT9. Multiple unpaired t-test (P value) followed by FDR (Q value) analysis (d, e, g–i). Mean, one-way ANOVA with Dunnett’s test (j). Mean ± s.e.m., two-way ANOVA with Sidak’s test (k–m). *(black), P < 0.05; *(cyan), P < 0.01; *(red), P < 0.001; ns, P > 0.05.
