Figure EV5. T‐loop autophosphorylation is critical for CDKL5 activity towards CEP131.

1. HEK293 cells were co‐transfected with untagged CDKL5 (wild type “WT” or the mutants indicated) and FLAG‐tagged CEP131 [wild type (WT), a Ser³⁵Ala mutant (SA)] or empty vector (−). Anti‐FLAG precipitates were subjected to Western blotting with the antibodies indicated. The input extracts were also subjected to immunoblotting (lower panels). Three independent experiments were done, and one representative experiment is shown.
2. Anti‐FLAG precipitates from HEK293 cells transiently expressing FLAG‐tagged CDKL5 (wild type “WT” or the mutants indicated) were incubated with a synthetic peptide corresponding to the sequence around the CEP131 Ser³⁵ phosphorylation site, in the presence of [γ‐³²P]‐labelled ATP‐Mg²⁺. Peptide phosphorylation was quantitated in a scintillation counter. Data are represented as mean ± SEM from three independent experiments.

Source data are available online for this figure.