Fig. 4

cIAP1 is a downstream protein molecule of TRIM56 in glioma. a Images of Human Ubiquitin Array (500 μg lysate, 2 min exposure) performed with lysates prepared from GBM#P3-NC and -sh-TRIM56 cells treated with 20 μg/mL of the proteasome inhibitor MG132 for 6 h before collection. Specific quantification of spots with significantly elevated levels of ubiquitination was performed and the two candidate spots are highlighted in the colored boxes. b Venn diagrams showing the intersection of genes that are differentially expressed in both the TCGA and CGGA databases and also show changes in the ubiquitin arrays for both GBM#P3 and LN229 cells in a. c Western blot analysis of two candidate target proteins, cIAP1 and A20, in GBM#P3-, LN229- and U118MG-shTRIM56-NC, -S1 and -S2 cells. d-e Expression levels of cIAP1 in glioma patient tissue and normal brain tissue based on TCGA-GBM, GSE108474, TCGA-LGG GBM, CGGA and Rembrandt datasets. f Representative images and quantification of immunostaining of cIAP1 in different grades of human glioma and normal brain tissue (NBT) samples (magnification: × 200, × 400). g Representative images of immunostaining for cIAP1 in sections from intracranial xenografts derived from GBM#P3- and LN229-shTRIM56-NC, -S1 and -S2 implanted in nude mice (scale bar, 100 µm). h Fluorescence images of immunofluorescence staining for TRIM56 and cIAP1 in different grades of human glioma and normal brain tissue samples (NBT) acquired using confocal microscopy (scale bar, 5 µm). i Fluorescence images of immunofluorescence staining for TRIM56 and cIAP1 proteins to determine subcellular localization in GBM#P3, LN229 and U118MG glioma cell lines. Immunofluorescence analysis was performed using specific antibodies as shown and images were acquired using confocal microscopy (scale bar, 40 µm). DAPI was used for staining nuclei. Comparisons between two independent samples and among multiple samples were performed using two-tailed t tests and one-way ANOVA, respectively. Error bars indicate at least three independent experiments, and data are shown as mean ± SEM. ∗ p < 0.05, ∗  ∗ p < 0.01, ∗  ∗  ∗ p < 0.001, and ∗  ∗  ∗  ∗ p < 0.0001