A quantitative phosphoproteomic approach for the identification of cellular CDKL5 targets A quantitative phosphoproteomic approach for the identification of cellular CDKL5 targets Pathogenic and non‐pathogenic CDKL5 variants. Schematic diagram shows modular domains in CDKL5 and the position of amino acid substitutions in humans that are either pathogenic, non‐pathogenic or of unknown consequence according to RettBASE (http://mecp2.chw.edu.au/cdkl5/cdkl5_variant_list_copy.php; Krishnaraj et al, 2017). NES: nuclear export signal; NLS: nuclear localization signal.Generation of CDKL5 knockout human cells. U2OS cells modified with the Flp‐In™ T‐REx™ system were subjected to genome editing to disrupt CDKL5. Extracts of cells from two different knockout (KO) clones (7 and 13) were subjected to SDS–PAGE on the gel types indicated followed by immunoblotting with in‐house anti‐CDKL5 antibodies. “Hi” higher exposure; “lo” lower exposure. Asterisk: non‐specific band.Stable expression of CDKL5 in knockout clone 13. The CDKL5 open reading frame (1,030 amino acid/115‐kDa isoform) was inserted at the FRT sites in CDKL5 knockout clone 13 from (B). Cells transfected with empty vector were used as control. Cells were incubated with the indicated concentrations of tetracycline (Tet), and extracts were immunoblotted with anti‐CDKL5 antibodies.Phosphoproteomics workflow. CDKL5 knockout clone 13 and the same cells re‐expressing CDKL5 were lysed and protein extracts were digested using trypsin. After phosphopeptide enrichment by TiO2 chromatography, peptides were isotopically labelled by TMT and combined. Combined peptides were fractionated by high‐pH reversed‐phase chromatography. Fractions were separated on a nano‐HPLC and analysed by quantitative mass spectrometry on an Orbitrap Fusion mass spectrometer. Data were analysed using MaxQuant software. Source data are available online for this figure. Ivan M Muñoz et al. EMBO J. 2018;embj.201899559 © as stated in the article, figure or figure legend