Figure 3. Ubiquitin-conjugating enzyme E2T (UBE2T) interacts with GRP78 and GRP78 is an independent risk factor for gliomas. (A) SDS-PAGE analysis of UBE2T immunoprecipitates. The indicated band was excised and examined via mass spectrometry. (B) The mass spectrometry result allowed the identification of proteins. (C) U251 cell lysates were incubated with Protein A/G Sepharose conjugated with anti-UBE2T and anti-GRP78 antibodies. The immunoprecipitates were detected via immunoblotting. (D) Immunoblotting of GRP78 protein levels in normal brain and GBM tissues. Expression levels were normalized to those of GAPDH. (E) Immunohistochemistry of GRP78 in human GBM tissues compared with that in normal brain tissues (×100). Scale bar, 100 μm. (F) Multivariate analysis of the relationship of GRP78 with overall survival (OS) in patients with glioma using the Chinese Glioma Genome Atlas RNA sequencing dataset. (G) Representative images of the immunohistochemical staining of UBE2T and GRP78 in glioblastoma (n = 43, ×100). Scale bar, 100 μm. (H) The correlation between UBE2T and GRP78 protein levels was analysed in glioblastoma tissues. The samples were classified into low and high expression groups based on the UBE2T and GRP78 IHC scores. The protein expression level of GRP78 was positively correlated with the GRP78 expression level. Each dot indicates the relative protein expression level. Data were analysed using Pearson’s correlation coefficient. (Error bars indicate the SEM of three independent experiments. Two-tailed Student’s t-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001).