Journal: Neural Regeneration Research

Article Title: The Citron homology domain of MAP4Ks improves outcomes of traumatic brain injury

doi: 10.4103/NRR.NRR-D-24-00113

Figure Lengend Snippet: MAP4Ks negatively regulate Wnt/β-catenin signaling. (A) Western blots showing CTNB1 destabilization by WT but not KM MAP4Ks. Cells were treated with OA for potential stimulation of kinase activity. ACTB: loading control. (B) Suppression of the β-catenin-responsive TOPFlash luciferase reporter by MAP4Ks (mean ± SEM; n = 3 per group; P = 0.0171 for HGK, P = 0.0015 for MINK1, and P = 0.0003 for TNIK). (C) Western blots showing CTNB1 stabilization through downregulation of MAP4Ks. JNK phosphorylation, a target of the non-canonical Wnt signaling, is not obviously altered. shLuc: a control for shRNA-mediated knockdown. (D) Enhanced activity of the β-catenin-responsive TOPFlash luciferase reporter by downregulation of MAP4Ks (mean ± SEM; n = 3 per group; P = 0.0022 for shHGK, P = 0.0004 for shMINK1, and P = 0.0002 for shTNIK). (E) Western blots showing CTNB1 stabilization by K02288-mediated inhibition of MAP4Ks. Cells treated with Wnt3A-conditioned medium (Wnt3A-CM) were used as positive controls. JNK phosphorylation was not obviously altered by K02288. (F) Co-immunoprecipitation results showing a lack of interaction between MAP4Ks with CTNB1. (G) A schematic diagram summarizing the functional regulation of the crosstalk between MAP4Ks and Wnt signaling by CNH. ACTB: Actin beta; CNH: Citron homology domain; CTNB1: β-catenin; GFP: green fluorescent protein; HA: hemagglutinin; KM: kinase-dead mutant; OA: okadaic acid; WT: wild-type.

Article Snippet: The following primary antibodies were used for western blot: FLAG (1:5000, Cat# 20543-1-AP, Proteintech, Rosemont, IL, USA), HA (1:5000, Cat# MMS-101P, Biolegend), phospho-threonine (p-Thr; 1:1000, Cat# 9386, Cell Signaling Technology, Danvers, MA, USA), actin beta (ACTB; 1:10000, Cat# HRP-66009, Proteintech), catenin beta 1 (CTNB1; 1:3000, Cat# 9386, Cell Signaling Technology), p-JNK (1:2000, Cat# 9251, Cell Signaling Technology), JNK (1:3000, Cat# A5005, Selleckchem, Houston, TX, USA).

Techniques: Western Blot, Activity Assay, Control, Luciferase, Phospho-proteomics, shRNA, Knockdown, Inhibition, Immunoprecipitation, Functional Assay, Mutagenesis