Fig. 4: Cdkl5 phosphorylates Sox9 at Serine 199 site.

a Bilateral renal ischemia was induced in C57BL/6 mice for 30 min followed by reperfusion for 1 day. Renal cortical lysates were then used to immunoprecipitate Cdkl5, while IgG was used as negative control. Immunoprecipitates were then run on a 4–12% gradient SDS-PAGE gel followed by protein visualization with SYPRO Ruby Protein Gel Stain. The ~65-kDa Cdkl5-interacting protein was then identified by mass spectrometric analysis as Sox9 as described in the “Methods” section. b Purified wild-type Cdkl5 and wild-type and mutant Sox9 proteins were co-incubated in a kinase assay buffer with [gamma-32P]-ATP for 30 min. Samples were then run on SDS-PAGE gel followed by transfer to PVDF membrane. Radiolabeled Sox9 was examined by autoradiography, followed by western blot analysis to examine the input proteins. Blots are representative of two independent experiments. c Schematic representation of Sox9 protein (modified from ref. ⁶⁴). Protein sequence analysis showed that the sequence surrounding the Ser-199 site is highly conserved. HMG indicates high-mobility group box DNA-binding domain, CD indicates conserved domain, and PQA indicates proline–glutamine–alanine-rich domain. d Control, cisplatin, and ischemic renal tissues from control and Cdkl5^PT−/y mice were subjected to immunoblot analysis of indicated proteins. Blots are representative of at least three independent experiments. e, f Densitometric analysis of Sox9 and p-Ser-199–Sox9 protein levels. The graph represents cumulative results (n = 5 independent biological samples) from three independent experiments. Densitometric analysis was carried out using Image J, and the signals of indicated proteins were normalized by actin levels in the same samples. In all the bar graphs, experimental values are presented as mean ± s.e.m. The height of error bar = 1 s.e., and p < 0.05 was indicated as statistically significant. One-way ANOVA followed by Tukey’s multiple-comparison test was carried out, and statistical significance is indicated by *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.