Fig. 5: PTPN13 interacts with IGF2BP1 through the fifth PDZ domain.

A Schematic diagram of the KIND, FERM, PTP, and five PDZ domains in the PTPN13 protein. B A CoIP assay was performed using an anti-PTPN13 antibody and IgG control antibody incubated with nuclear extracts of PLC/PRF/5 cells with a transient overexpression vector or a control vector, followed by silver staining. A red arrow indicates IGF2BP1. C Left: Interacting proteins based on mass spectrometry after silver staining in the CoIP experiments described in (B) in two pairs of transient overexpression cells (PLC/PRF/5-OE vs empty and LM3-OE vs empty) and three pairs of stable knockdown cells (Huh7-sh vs control, SK-hep1-sh vs control, and SMMC-7721-sh vs control). Right: A Venn diagram of protein spectra analysis results and overexpressed mRNAs in the TCGA cohort, which identified four key PTPN13-interacting genes. D Immunoblot analysis showed the specific association of IGF2BP1 with PTPN13. Twenty-four hours after transfection, whole-cell lysates were prepared and subjected to immunoprecipitation with an anti-PTPN13 antibody, followed by immunoblotting with an anti-IGF2BP1 antibody. GAPDH was used as a negative control for PTPN13-interacting proteins. E Mammalian two-hybrid assay. Plasmids encoding PTPN13 domains (pBIND-domain) and IGF2BP1 (pACT-IGF2BP1) and the pG5luc vector were cotransfected into HEK293T cells. The pBIND-domain plasmids were further used to examine which part of PTPN13 was involved in the physical interaction. pACT-MyoD and pBIND-Id were used as positive controls. Data were normalized to Renilla reniformis luciferase activity. F CoIP assay and immunoblot analysis of Flag-tagged PTPN13 (domain truncation fragments) immunoprecipitated by HA-tagged IGF2BP1. The data represent the mean ± SD of three independent experiments (***P < 0.001).