Fig. 4: Ankyrin-G interacts with OTUD7A and its levels are decreased in Df(h15q13)/+ mouse brain. | Molecular Psychiatry

Fig. 4: Ankyrin-G interacts with OTUD7A and its levels are decreased in Df(h15q13)/+ mouse brain.

From: Impaired OTUD7A-dependent Ankyrin regulation mediates neuronal dysfunction in mouse and human models of the 15q13.3 microdeletion syndrome

Fig. 4

a STRING analysis of functional network interactions for Ankyrin-G-190-BioID2 (from 3 biological replicates). Green nodes represent proteins shared with the OTUD7A-BioID2 list. b Venn diagram of shared protein interactors between Ankyrin-G-190 and OTUD7A. c Co-immunoprecipitation of OTUD7A-FLAG with Ankyrin-G-190-EGFP and d Ankyrin-B-2XHA from co-transfected HEK293 FT cells. e Co-immunoprecipitation of endogenous 3XFLAG-OTUD7A with Ankyrin-G and Ankyrin-B in P14 C57BL/6-3XFLAG-OTUD7A mouse cortex. f Schematic of mouse Ankyrin-G showing protein domains. g Domain mapping of Ankyrin-G-OTUD7A interaction from co-transfected HEK293 FT cells expressing HA-Ankyrin-G domains and OTUD7A-FLAG or h the catalytic domain of OTUD7A (OTUD7A183-449-FLAG). i Representative image of DIV 14 C57BL/6J-3XFLAG-OTUD7A mouse cortical neurons stained for Ankyrin-G, FLAG, and MAP2. 63× objective; Top, scale bar = 20 μm; bottom (Inset zoom), scale bar = 5 μm. j Representative western blot and k protein levels of Ankyrin-G-190 in P14 cortex from WT and Df(h15q13)/+ mice; WT: n = 10 cortices, Df(h15q13)/+: n = 11 cortices; **p < 0.01; Student’s t-test, t = 3.070, df = 19. l Representative SIM images from DIV 17 WT and Df(h15q13)/+ cortical neurons; Scale bar = 5 μm. m Spine morphology analysis. n = 13 WT and 18 Df(h15q13)/+ cortical neurons (one dendrite per neuron). *p < 0.05, Unpaired t-test (two tailed); Mushroom: t = 2.301, df = 29, p = 0.0288; Thin: t = 0.4023, df = 29, p = 0.6904; Stubby: t = 0.9324, df = 29, p = 0.3588. n Mushroom spine head. n = 322 spines WT, n = 350 spines Df(h15q13)/+. ****p < 0.0001, Mann–Whitney test two-tailed (approximate), U = 40466; WT: median = 0.4975, Df(h15q13)/+: median = 0.3680. o Proportion of Ankyrin-G (+) mushroom spines. **p < 0.01, Student’s unpaired t-test; t = 3.196, df = 29. p Ankyrin-G puncta number in mushroom spine heads. n = 322 spines WT, n = 350 spines Df(h15q13)/+. ****p < 0.0001, Mann–Whitney test two-tailed (approximate), U = 44355; WT: median = 1, n = 322; Df(h15q13)/+ median = 0, n = 350. q Ankyrin-G total nanodomain area in mushroom spines. Mann–Whitney test two-wailed (approximate), p = 0.2204, U = 18693; WT: median = 0.046, n = 244 spines; Df(h15q13)/+: median = 0.052, n = 165 spines. r Ankyrin-G puncta density in dendrites. ***p < 0.001, Mann–Whitney test two-tailed, p = 0.004, U = 32; WT: median = 1.548, n = 13; Df(h15q13)/+: median + 0.8897, n = 18. s Ankyrin-G puncta size in dendrites. Values are the average puncta size per dendrite. Mann–Whitney test two-tailed (Exact), p = 0.079, U = 73; WT: median = 0.03100, n = 13; Df(h15q13)/+: median = 0.02750, n = 18.

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