Figure 8

Figure 8 CDKL1 function in cell signaling.

(A) CDKL1 variants increase signaling via p38 (MAPK8). Total lysates of HEK293T cells transiently expressing CDKL1^WT, CDKL1^Thr135Met, CDKL1^Cys143Arg, or CDKL1^Ser206Leu were cultured under basal conditions (DMEM containing 10% serum), lysed, and subjected to immunoblotting as indicated. Cells expressing kinase-dead CDKL1^Lys33Arg and cells transfected with empty vector (EV) were used as controls. GAPDH was used as loading control. Representative immunoblots from 5 independent experiments (n = 5) are given. Graph shows means (± SD) of the relative phosphorylation of p38 (at Thr¹⁸⁰ and Tyr¹⁸²) normalized to amounts of total p38 as well as to GAPDH. Phosphorylation levels in cells expressing CDKL1 mutants are relative to those in CDKL1^WT cells. One-way ANOVA with Tukey’s multiple-comparison test was used. *P ≤ 0.05, **P ≤ 0.01. (B) Knockdown of Cdkl1 induces Vegfaa expression. Quantitative PCR analysis was performed on total RNA of 24 hpf zebrafish embryos. Graphs show amounts of axin2, patched1, and vegfaa transcripts in Cdkl1 splMO relative to CTRL MO–treated embryos. n = 8 experiments; mean values (red bars) and individual experiments (circles) are indicated. **P = 0.07, 2-tailed Wilcoxon’s test. (C) Expression of the arterial marker ephrin B2a (ephB2a) and of plexin D1 (plxnD1) is stronger in Cdkl1 morphants than in control-injected embryos. Spatial expression was determined by in situ hybridization. Stacked bar graphs summarize 3 experiments with 82 (CTRL MO) and 85 embryos (Cdkl1 splMO) for ephB2a and 3 experiments with 63 (CTRL MO) and 42 embryos (Cdkl1 splMO) for plxnD1. **P = 0.0018, ***P = 0.0003, 2-sided Fisher’s exact test. All experiments were done at 24 hpf. Scale bars: 200 μm.