Figure 6

Disease-associated CDKL1 variants show different protein-protein interaction profiles.

EGFP-tagged CDKL1 protein variants were expressed in HEK293T cells, purified by GFP-Trap, and subjected to liquid chromatography–tandem mass spectrometry analysis. Experimental duplicates were performed for all conditions. Protein abundances were normalized based on GFP protein peptide abundances. (A and B) Heatmaps: Pearson correlation–based hierarchical clustering of EGFP-vector, CDKL1^WT, CDKL1^Lys33Arg, CDKL1^Thr135Met, CDKL1^Cys143Arg, and CDKL1^Ser206Leu samples with average linkage, based on 626 ANOVA-significant proteins (A) and on 36 cilium proteins, assigned to the GOCC gene set Cilium (B) between all analyzed phenotypes (adjusted P value < 0.05). Normalized protein abundances were scaled before clustering for visual purposes. Relative protein abundance is coded by colors from red (high abundance) to blue (low abundance). (C and D) Validation of differentially affected protein-protein interactions. Total cell lysates (TCL) of HEK293T cells expressing EGFP-tagged CDKL1 protein variants or EGFP (control) were subjected to immunoprecipitation (IP) using GFP-Trap beads. CDKL1 expression and precipitation efficiencies were determined by anti-GFP immunoblotting. Coprecipitation and input levels of endogenous IFT52, IFT172, CFAP20, and TUBA1A were assessed by immunoblotting using specific primary antibodies; GAPDH was used as loading control (C). The graph shows mean relative amounts of coprecipitated IFT52, IFT172, CFAP20, and TUBA1A normalized to precipitated GFP (D). The mean of experiments for CDKL1^WT (IFT52, IFT172, CFAP20) and CDKL1^Ser206Leu (TUBA1A) was set to 1. **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, 1-way ANOVA. n = 3 experiments.