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. 2019 Oct 15;14(10):e0223790. doi: 10.1371/journal.pone.0223790

Fig 2. Expression of STUB1DN mutants leads to the formation of protein aggregates.

Fig 2

ARPE-19 cells were transduced using lentiviral particles containing vectors for the expression of either STUB1K30A or STUB1H260Q. Control cells were transduced with empty vector. Cells were further incubated in the presence or absence of 10uM of MG-132 and 50nM of BafA for 6h. A) Cell extracts of proteins insoluble in increasing concentrations of SDS where filtered through a nitrocellulose membrane and trapped proteins were blotted with antibodies directed against ubiquitin, p62 and myc tag. Expression of STUB1-DN mutants increases the amount of protein aggregates trapped in the membrane both in the presence or absence of MG-132 or BafA. B) Western blotting for the proteins present in the supernatant (soluble proteins) shows a decrease in the levels of ubiquitin conjugates in the STUB1-DN mutant expressing cells (compare lane 1 with lane 4 and 7). All samples were analyzed under the same experimental conditions. The results represent the mean ±SD of N = 3 independent experiments (n.s. nonsignificant; *p < 0.05; **p < 0.01; ***p < 0.001).