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Interaction with TBC1D23 is required for LKB1 to promote neuronal development and brain growth. a HEK293T cells were transfected with indicated constructs, and then immunoprecipitated with GFP beads and probed with indicated antibodies. The graph showed ratios of bound TBC1D23 to GFP quantified by densitometry. b MBP-LKB1 CRD-LFa WT, mutants, or MBP pull-down of purified GST-TBC1D23 PH domain. Red arrow indicates GST-TBC1D23 PH. c Affinity between TBC1D23 PH domain and LKB1-LFa WT or mutants, determined by ITC. Mean association constants (Ka) are shown, with error bars indicating SD from 3 independent titrations. d Morphology of CaP axons in Tg[Hb9:GFP]ml2 transgenic zebrafish at 48 hpf with z-axis scanning, scale bar, 50 μm. Lateral views (upper) and enlarged views of rectangles (lower) are shown. e Statistical analysis of the length of CaP axons in zebrafish at 48 hpf. Approximately 6 to 11 Tg[Hb9:GFP] transgenic zebrafish embryos from each group were measured for 3 CaP axons at approximately the third to fifth CaP axon located on the yolk extension, with each point representing the average axon length of one embryo. f HuC (green) expression in Tg[HuC:GFP] transgenic zebrafish at 48 hpf. Top, lateral view; bottom, dorsal view. scale bar, 200 μm. g The size of zebrafish midbrain at 48 hpf. The overview fluorescent images are presented as maximum intensity z-projections. White arrowheads point to the midbrain. FB, forebrain; MB, midbrain; OT, optic tectum; CB, cerebellum; HB, hindbrain; SC, spinal cord; E, eye. The size of the midbrain was measured from the lateral view, and 8 to 21 embryos from each group were used for comparison. Results are presented as mean ± SD (a–g), and p values were calculated using one-way ANOVA, Dunnett’s multiple comparisons test (a and e) or by Kruskal–Wallis test (g). Experiments were repeated 3 times (a–g). h Simplified model depicting how TBC1D23 and LKB1 cooperate to promote AMPK activation at Golgi and to regulate neuronal development. TBC1D23 mutation/deletion or LKB1 mutation/deletion impairs the recruitment of LKB1 to Golgi, leading to compromised Golgi-AMPK activation, aberrant neuronal development, and eventually pontocerebellar hypoplasia (PCH). Source data are provided as a Source data file.
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