Fig 2: Assigning interaction of praja2 with other proteins possibly related to GBM.a Praja2 protein–protein interaction (PPI) network. Praja2 complexes were purified from cell lysates of HEK293 cells transiently transfected with Flag-praja2rm vector or flag vector (control), which were independently subjected to an enrichment step with an anti-Flag derivatized resin, whose eluates were finally subjected to proteomic analysis. Identified praja2-binding partners involved in metabolic pathways and those reported in available databases were used to generate a PPI network. Connections are colored based on the interaction source: interactors identified in this study (green edges), CPTAC-GBM interactors (red edges), Intact interactors (blue edges), and STRING interactors (gray edges). The complete list of protein interactors is reported in Supplementary Data 1. b Schematic representation of praja2 constructs (lower panel) used in co-immunoprecipitation assays (upper panel). Lysates from HEK293 cells transiently transfected with Myc-tagged KSR2 and Flag-praja2 vectors were subjected to immunoprecipitation with an anti-Myc antibody. Precipitates and an aliquot of lysates were immunoblotted with anti-Flag and anti-Myc antibodies. c In vitro translated, [35S] labeled KSR2 was subjected to pull-down assay with GST and GST-praja2 polypeptides. d U87MG cells were immunostained with anti-Myc and anti-praja2 antibodies and further analyzed by confocal microscopy. Scale bar = 5 µm. The Pearson’s coefficient value of KSR2 and praja2 signals is shown (lower, right panel). e A trimeric complex composed of praja2, KSR2, and AMPKa1 was isolated from lysates of HEK293 cells transiently expressing Flag-praja2 and KSR2-Myc and subjected to immunoprecipitation with anti-Flag. f HEK293 cells transfected with HA–ubiquitin and Flag-praja2 or Flag-praja2rm and KSR2-Myc were treated for 6 h with 10 µM MG132. Lysates were immunoprecipitated with an anti-KSR2 antibody. Lysates and precipitates were immunoblotted with anti-HA, anti-Flag, and anti-KSR2 antibodies. g Lysates from growing HEK293 cells transiently transfected with HA–ubiquitin, KSR2-Myc, and control siRNA (siRNAc) or siRNAs targeting praja2 (siPraja2) were immunoprecipitated with anti-KSR2 antibody. Lysates and precipitates were immunoblotted with anti-HA, anti-praja2, and anti-KSR2 antibodies. h Cells were transiently transfected with flag-praja2 or flag-praja2rm for 24 h. Lysates were immunoblotted with the indicated antibodies. i Quantitative analysis of data shown in panel h. A mean value ± SEM of three independent experiments is reported. P value: * = 0.037; ** = 0.0026. j U87MG cells were transfected with a control vector or with vectors encoding for flag-praja2 or flag-praja2rm. Where indicated, cells were pretreated with MG132 for 6 h before harvesting. Lysates were subjected to immunoblot analysis with the indicated antibodies. k Quantitative analysis of data shown in panel j. A mean value ± SEM of four independent experiments is reported. P value: * = 0.011; ** = 0.0020.