Fig. 2.
CDK5 binds to and phosphorylates TRIM59 at S308. a Immunoprecipitation (IP) and WB for CDK5 binding to TRIM59 in LN229/EGFRvIII and U87/EGFRvIII cells. b IP and WB for CDK5 binding to TRIM59 in LN229/EGFR and U87/EGFR cells with or without EGF stimulation. c In vitro GST pull-down analysis. Purified GST-CDK5 or GST proteins were mixed with cell extracts from U87/EGFR cells with or without EGF stimulation for 30 min. d Schematics of TRIM59 WT and truncated constructs. e CDK5 interacts with TRIM59 with amino acid residues 281–403. TRIM59 WT or indicated mutants was co-transfected into LN229/EGFR cells with Flag-tagged CDK5 followed by EGF stimulation. f Effect of CDK5 knockout on p-Ser/Thr (p-S/T) of TRIM59 in LN229/EGFR GBM cells with EGF stimulation. g Effects of mutations of TRIM59 T144A, S246A, and S308A on TRIM59 phosphorylation in LN229/EGFR cells expressing Flag-CDK5 with EGF stimulation. h In vitro kinase assays of recombinant active CDK5/p35 with purified TRIM59 WT or S308A mutant as a substrate. The reaction samples were assessed by WB performed with anti-p-TRIM59S308, anti-TRIM59, anti-CDK5, or anti-p35 antibody. i Localization of TRIM59 WT, S308A, and S308D mutant in LN229/EGFR cells. S308D, a constitutive phosphorylated S308 mutant. j IF analysis for TRIM59 WT, S308A, and S308D mutant in LN229/EGFR cells with or without EGF stimulation. Scale bar, 40 μm. Data are representative of three independent experiments with similar results. Source data are provided as a Source Data file