Schematic illustration of the potential mechanisms by which MIG‐6 regulates GLUT1 gene expression.
Immunoblotting analysis for HIF1α protein expression in BT549 cells with GFP or MIG‐6 knockdown.
Immunoblotting analysis for HIF1α protein expression in MDA‐MB‐231 cells with GFP or MIG‐6 knockdown.
Real‐time PCR analysis for HIF1α mRNA expression in BT549 cells with GFP or MIG‐6 knockdown. The quantified results are presented as mean ± SD (n = 4). ***P < 0.001, by Student's t‐test.
Immunoblotting analysis for HIF1α protein expression in GFP and MIG‐6 knockdown BT549 cells in the absence or presence of the proteasome inhibitor MG132.
GFP and MIG‐6 knockdown BT549 cells transfected with the indicated plasmids were treated with MG132, subjected to immunoprecipitation (IP) with Flag‐tag or HIF1α antibody, followed by immunoblotting analysis. MG132 was used to rescue HIF1α protein degradation mediated by MIG‐6 knockdown, leading to similar HIF1α protein levels in control and MIG‐6 knockdown BT549 cells, which were used as the input to examine the role of MIG‐6 in the interaction between HIF1α and HAUSP.
Luciferase and MIG‐6 knockdown 293 cells were transfected with the indicated plasmids and subjected to MG132 treatment. Afterward, cells were harvested for IP with HA antibody, followed by immunoblotting analysis to determine the level of K48‐linked ubiquitination of HIF1α. MG132 was used to preserve the degradative K48‐linked ubiquitination signals.
Immunoblotting analysis for MIG‐6 and HIF1α expression in Luciferase and MIG‐6 knockdown 293 without MG132 treatment.
Immunoblotting analysis for GLUT1 protein expression upon MIG‐6 overexpression in GFP and HIF1α knockdown BT549 cells.
Immunoblotting analysis for GLUT1 protein expression upon MIG‐6 overexpression in GFP and HAUSP knockdown BT549 cells.
Immunoblotting analysis for HIF1α protein expression upon HAUSP overexpression in GFP and MIG‐6 knockdown BT549 cells.
