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. 2020 Mar 8;7(8):1902040. doi: 10.1002/advs.201902040

Figure 2.

Figure 2

OTUD6B negatively regulates HIF pathway. A) Immunoblotting of HIF‐1α, HIF‐2α and OTUD6B in MHCC‐LM3 cells transfected with indicated shRNA. B) Q‐PCR was used to examine the mRNA level of HIF target genes in MHCC‐LM3 cells transfected with indicated shRNA. C) HA‐Ub was cotransfected together with indicated shRNA into HEK293T cells. Cells were treated with MG132 for 8 h before collection. Then HA‐Ub was immunoprecipitated with anti‐HA antibody and immunoblotted with anti‐HIF‐1α antibody. D) Transwell assay was performed in HCC cells transfected with indicated shRNA (scale bar, 50 µm). E) The means of migrated cells in (D) were shown as the column chart. F) Immunoblotting of HIF‐1α, HIF‐2α, and OTUD6B in SMMC7721 cells with stable OTUD6B overexpression. G) Q‐PCR was used to examine the mRNA level of HIF target genes in SMMC7721 cells with stable ectopic OTUD6B overexpression. H) HA‐Ub was cotransfected together with indicated constructs into HEK293T cells. Cells were treated with MG132 for 8 hours before collection. Then HA‐Ub was immunoprecipitated with anti‐HA antibody and immunoblotted with anti‐HIF‐1α antibody. I) Transwell assay was performed in HCC cells transfected with indicated constructs (scale bar, 50 µm). J) The means of migrated cells in (I) were shown as the column chart. K) The purified His‐pVHL or His‐HIF‐1α was incubated respectively with purified GST or GST‐OTUD6B. The mixtures were subjected to GST pull down and blotted. Results are shown as mean ± s.d. n = 3 independent experiments. * P < 0.05, ** P < 0.01 compared with NC group, # P < 0.05, ## P < 0.01 compared with group of OTUD6B knockdown or ectopic OTUD6B overexpression, Student's t test.