Fig 1: EB2 phosphorylation by ICK and CDKL2 in HEK293T cells and primary neurons.A CDKL5 and its closely-related kinases on the human phylogenetic tree. B Western blot showing levels of FLAG, total EB2 and EB2 pS222 in HEK293T cells co-transfected with StrepII-tagged EB2 and FLAG-tagged ICK WT, MAK WT, MOK WT, CDKL1 WT, CDKL2 WT, CDKL3 WT, CDKL4 WT, CDKL5 WT or CDKL5 kinases-dead (KD). C Western blot showing levels of FLAG, total EB2 and EB2 pS222 in HEK293T cells co-transfected with StrepII-tagged EB2 and FLAG-tagged ICK WT/KD, CDKL2 WT/KD or CDKL5 WT/KD. D–F EB2 pS222 quantification comparing WT and KD of ICK, CDKL2 and CDKL5 expressing cells respectively. n = 6 per condition. Mann–Whitney test. **p ≤ 0.01. G DIV8 Cdkl5 KO primary mouse hippocampal neurons, overexpressing FLAG-tagged ICK, CDKL2 or CDKL5 WT, were co-stained with FLAG, EB2 pS222, total EB2 and DAPI. Full lines highlight neurons expressing the kinases and dashed lines highlight neurons which do not express the kinases. Scale bar is 30 μm. H Quantification of EB2 pS222 mean fluorescence in Cdkl5 WT neurons, in Cdkl5 KO neurons and in Cdkl5 KO neurons overexpressing the three kinases. n = 21–166 per condition. Mann–Whitney test. ****p ≤ 0.0001. I Quantification of total EB2 mean fluorescence in Cdkl5 WT neurons, in Cdkl5 KO neurons and in Cdkl5 KO neurons overexpressing the three kinases. n = 21–166 per condition. Mann–Whitney test. ****p ≤ 0.0001.
Fig 2: WDHD1 forms a complex with MAPRE2 and increases the ubiquitination degradation of MAPRE2. (A) Western blot results indicating that WDHD1 expression was higher in A549/DDP cells than in A549 cells. MAPRE2 was upregulated, and DSTN did not significantly change after knocking out WDHD1. (B,C) The results from an MTT assay and flow cytometry showing that after MAPRE2 was knocked out in A549 cells, the drug sensitivity of lung adenocarcinoma cells to cisplatin decreased, viability increased, and apoptosis decreased. (D) Coimmunoprecipitation showing that WDHD1 and MAPRE2 are interacting proteins. (E) Immunofluorescence demonstrating that WDHD1 (red) is primarily present in the nucleus, and MAPRE2 (green) is present in both nucleus and cytoplasm. (F) Protein stability experiments confirming that MAPRE2 degradation slowed down after knocking out WDHD1. DDP: IC25 concentration of cisplatin for A549/DDP cells. n = 3; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Fig 3: Screening of CDKL5 inhibitors in rat primary neurons using western blotting.Western blots showing expression of total EB2 and levels of Ser222 EB2 phosphorylation in DIV14-16 rat primary neurons upon treatment of 1 hr with 5 nM, 50 nM, 500 nM, and 5000 nM of selected CDKL5 inhibitors. Figure 1—source data 1.Source data contains raw blots.
Fig 4: CDKL2 phosphorylates CDKL5 substrates in mouse brain.A Schematic of the generation of the dual Cdkl5/Cdkl2 KO mouse model. An insert of 700 bp containing the exon 4 of Cdkl2 was deleted from Cdkl5 KO embryos using CRISPR-Cas9. B Western blot showing levels of CDKL5, total EB2 pS222, tubulin and CDKL2 in the cortex of P10 dual Cdkl5/Cdkl2 KO animals. The arrow indicates CDKL2, [*] indicates a non-specific band. C Quantification of EB2 pS222 levels in the cortex of P10 Cdkl5/Cdkl2 KO animals. n = 3 animals per genotype with 2 technical replicates. Mann–Whitney test. **p ≤ 0.01. D Western blot showing levels of total MAP1S and MAP1S pS812 in the cortex of P10 dual Cdkl5/Cdkl2 KO animals. E Schematic encapsulating our findings. CDKL5 and CDKL2 phosphorylate EB2 on residue S222, a process modulated by calcium entry through NMDA receptors. Additionally, the dephosphorylation of EB2 S222 is attributed to the phosphatases PP1 and PP2A.
Fig 5: Inhibition of PP1 and PP2A increases EB2 phosphorylation in presence and absence of CDKL5.A Western blots showing CDKL5, total EB2 and tubulin expression and EB2 pS222 level upon 1 μM okadaic acid (OA) for 40 min, 20 μM cyclosporin A (CSA) for 40 min and 50 μM NMDA treatment for 20 min in DIV14-15 rat primary cortical culture. B Quantification of EB2 pS222 level upon 1 μM OA for 40 min, 20 μM CSA for 40 min and 50 μM NMDA treatment for 20 min in DIV14-15 rat cortical primary culture. Mann–Whitney test. n = 5 with 2 technical replicates per treatment. ****p < 0.0001. **p ≤ 0.01. *p ≤ 0.05. C Western blots showing total EB2 and tubulin expression and EB2 pS222 level upon 1 μM OA treatment for 40 min, 50 μM NMDA treatment for 20 min and a combination of OA and NMDA treatments (1 μM OA treatment for 20 min followed by 20 min of OA + NMDA) in DIV8 mouse Cdkl5 WT and KO primary cortical culture. D Quantification of EB2 S222 phosphorylation level upon 1 μM OA treatment for 40 min, 50 μM NMDA treatment for 20 min and a combination of OA and NMDA treatments in DIV8 mouse Cdkl5 WT and KO primary cortical culture. Mann–Whitney test. n = 3 with 2 technical replicates per treatment. **p ≤ 0.01. *p ≤ 0.05. Only significance within WT treatments and within KO treatments are shown. E Pairwise quantification of EB2 S222 phosphorylation upon 1 μM OA treatment for 40 min in Cdkl5 KO primary cortical culture. Mann–Whitney test. n = 3 with 2 technical replicates per treatment. *p ≤ 0.05.
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