Fig. 4: FBXO22 promotes HIF-1α and VEGFA expression by directly mediating VHL ubiquitin degradation.
From: FBXO22 promotes glioblastoma malignant progression by mediating VHL ubiquitination and degradation
A–C The protein expression of VHL in U373-shNC/shFBXO22, LN229-shNC/shFBXO22, and U87-Vector/FBXO22 cells was detected. D, E VHL mRNA expression in FBXO22 knockdown cells and FBXO22 overexpression cells was assessed by qRT-PCR assay. F, G After treatment with CHX, VHL protein expression was detected in FBXO22 overexpression, and the relative intensity of VHL protein was quantified using the software ImageJ. H After treatment with MG132 for 4 h, VHL protein expression in U87-Vector/FBXO22 cells was detected by western blots. I Western blots analysis of FBXO22 binding to VHL after IP HA in U87 after overexpression of HA-FBXO22 and Flag-VHL. J IP Flag was performed in 293T cells after transfection of the missing mutant with the corresponding domain, and Western blots analysis was used to detect the domain of VHL and FBXO22 interaction. K According to the website of Uniport (www.uniport.org), four missing bodies, as shown in the figure are constructed. L Western blots detection of VHL-associated ubiquitination after IP Flag in HA-FBXO22 and Flag-VHL overexpression cells. M HA-Ub-K48 and HA-Ub-K63 plasmids alone or cotransfected with HA-FBXO22 plasmid and Flag-VHL plasmid into U87 cells for 48 h, and the cells were then harvested and subjected to Flag IP. All the results were confirmed by three times repeated experiments. Statistical analysis was performed using unpaired t-tests. All statistical tests were two-sided. ns, no significance.
