Fig. 5: Complicated interactions exist between TRAF6 and IRAK1.

A, B Western blotting was used to detected the expression of TRAF6 and IRAK1 at protein levels in U87MG cells with miR-146a-5p overexpression, and TRAF6 or IRAK1 knockdown, and TRAF6 or IRAK1 overexpression. C The expression correlations of TRAF6 and IRAK1 at protein levels in GBM (n = 99) and normal brain tissue (n = 10) were analyzed in CPTAC databases. D Slides derived from two GBM samples were evaluated with TRAF6 (green) and IRAK1 (red) dual-labeled immunofluorescence (IF). H&E staining was used to evaluate the pathological features and to identify specific areas (adjacent area not the same zone). Left panel showed the microvascular proliferation area and right panel showed the leading edge (scale bar = 20 μm). The scale bar of the merged panoramic image is 2000 μm. E The expressions of TRAF6, IRAK1, and miR-146a at mRNA levels in different GBM areas were analyzed with Ivy GAP database. F The levels of TRAF6 and IRAK1 in GBM (n = 4) and LGG (grade II and III gliomas, n = 8) clinical samples were determined by IF staining and positive scores were of each sample based on the fluorescence intensities and numbers of positive cell. The positive scores of TRAF6 and IRAK1 in GBM were remarkably higher than those in LGG. G The Person correlation was used to analyze the correlation of TRAF6 and IRAK1 expressions in clinical samples at protein levels with the positive scores. H BiFC was used to estimate the spatial formation of the TRAF6-IRAK1 complex. When pBiFC-VN173-TRAF6 and pBiFC-CC155-IRAK1 or pBiFC-VN173-IRAK1 and pBiFC-CC155-TRAF6 were cotransfected into U87MG cells, green fluorescence was detected in the cytoplasm. Scale bar = 50 µm. I Coimmunoprecipitation (CoIP) was performed to evaluate interactions between TRAF6 and IRAK1 at the protein level. The anti-IRAK1 antibody immunoblotted (IB) the TRAF6 that immunoprecipitated (IP) from total protein, while the IRAK1 IP from cell lysates and IB with an anti-TRAF6 antibody.