Journal: Science advances

Article Title: The autism susceptibility kinase, TAOK2, phosphorylates eEF2 and modulates translation.

doi: 10.1126/sciadv.adf7001

Figure Lengend Snippet: Fig. 2. TAOK2β associates with the polyribosome complex, and its deficiency enhances general translation and protein synthesis. (A) Immunoblots of polysome fractions showing the presence or absence of TAOK2β across all polysome fractions isolated from Taok2+/+ (top) or Taok2−/− (bottom) mouse cortex. PABP1, RPL7a, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as positive and negative controls, respectively, to prove polysome preparation efficiency. (B) Polysome profiles from Taok2+/+ and Taok2−/− mouse cortices. (C) Quantification of polysome profiles in Taok2−/−, Taok2+/−, and Taok2+/+ mice cortices. Taok2+/+, n = 13; Taok2+/−, n = 8; Taok2−/−, n = 12; scanning electron microscopy (SEM) error bars, ordinary one-way analysis of variance (ANOVA) (P = 0.0251) followed by Dunnett’s multiple com- parisons test (*P < 0.05). (D) Immunostainings of acute cortical slices from the prefrontal cortex of Taok2+/+ and Taok2−/− mice analyzed by SUnSET assay. WT slices pre- treated with the translation inhibitor cycloheximide (WT + CHX) verifies the specificity of the puromycin antibody signal. (E) Immunoblot from acute cortical slices analyzed by SUnSET assay. (F) Quantification from (E). Taok2+/+, n = 5; Taok2+/−, n = 6; Taok2−/−, n = 5; SEM error bars, ordinary one-way ANOVA (P = 0.0167) followed by Dunnett’s multiple comparisons test (*P < 0.05). (G) Immunostainings of primary neurons (7DIV) analyzed by SUnSET assay. Cultured WT neurons were pretreated with CHX (WT + CHX). (H and I) Quantifications of immunofluorescent SUnSET assay of 7DIV neurons or 21DIV. 7DIV cells: Taok2+/+, n = 236 (five embryos); Taok2+/−, n = 113 (four embryos); Taok2−/−, n = 166 (four embryos). 21DIV cells: Taok2+/+ (three embryos), n = 81; Taok2+/−, n = 73 (two embryos); Taok2−/−, n = 79 (three embryos). SEM error bars, ordinary one-way ANOVA (P < 0.0001) followed by Dunnett’s multiple comparisons test (****P < 0.0001). Scale bars, 10 μm [(D) and (G)].

Article Snippet: The following antibodies were used in these studies for immunocytochemical or WB analysis: rabbit anti- Taok2β (Synaptic Systems, 395003; WB, 1:1000; Immunocytochemistry (ICC), 1:250; IP, 1:100); mouse anti- puromycin, clone 12D10 [Millipore, MABE343; WB, 1:10,000; immunohistochemistry (IHC); ICC, 1:5000]; chicken anti- MAP2 (Synaptic Systems, 188006; IHC, 1:500); guinea pig anti–β3- tubulin (Tuj1) (Synaptic Systems, 302304; ICC, 1:500); mouse anti–β- actin (Sigma- Aldrich, A5316; WB, 1:2000); rabbit anti–β- actin (13E5) (Cell Signaling Technology, 4970; WB, 1:5000); rabbit anti–Myc- tag (71D10) (Cell Signaling Technology, 2278; WB, 1:1000; ICC, 1:300); rabbit anti–immunoglobulin G (IgG) (Millipore, 03- 241; IP, 1:200); rabbit anti–phospho- eEF2 (Thr56) (Cell Signaling Technology, 2331; WB, 1:1000); rabbit anti–GSTtag (Cell Signaling Technology, 2625; WB, 1:1000); mouse anti– His- tag (27E8) (Cell Signaling Technology, 2366; WB, 1:1000); rabbit anti–His- tag (Cell Signaling Technology, 2365; WB, 1:1000); mouse anti- eEF2, clone 4B3- G7- H5 (Abcam, ab131202; WB, 1:2000); rabbit anti–phospho- eEF2K (Ser366) (Cell Signaling Technology, 3691; WB, 1:1000); rabbit anti- RPL7a (E109) (Cell Signaling Technology, 2415; WB, 1:1000); rabbit anti- PABP1 (Cell Signaling Technology, 4992; WB, 1:1,000); and mouse anti- eEF2K antibody (C- 12) (sc- 390710; WB, 1:200).

Techniques: Western Blot, Isolation, Electron Microscopy, Cell Culture