Fig. 5: MiR-375 inhibits UBE3A-mediated ubiquitination and degradation of DUSP1.

A, B SK-MES-1 and NCI-H2170 cell lysates were subjected to immunoprecipitation with negative control IgG, A anti-UBE3A or B anti-DUSP1 antibodies. The immunoprecipitates were then detected using the indicated antibodies. C Immunofluorescence analysis was used to detect subcellular colocalization of UBE3A and DUSP1, magnification ×600. D Western blotting was conducted to determine the expression of UBE3A in four LUSC cell lines and HBE cells. E, F The efficiency of UBE3A inhibition in SK-MES-1 cells and overexpression in NCI-H2170 cells, as determined by qRT‒PCR (E) and western blotting (F). G, H Western blotting and qRT‒PCR were carried out to verify the protein and mRNA levels of DUSP1 in UBE3A-depleted or UBE3A-overexpressing cell lines. I SK-MES-1 and NCI-H2170 cells were transfected with the indicated vector. After 48 h, the cell lysates were subjected to western blot analysis. The selected cells were treated with or without 10 µM MG132 for 12 h before being harvested. J SK-MES-1 and NCI-H2170 cells were transfected with the indicated vector. After 48 h, the cells were treated with 100 μg/mL CHX and collected for western blot analysis at the indicated time points. K SK-MES-1 and NCI-H2170 cells were transfected with the indicated plasmids. After 48 h, cell lysates were used to test the polyubiquitination level of DUSP1. The selected cells were treated with 10 µM MG132 for 12 h before being harvested.