Figure 5.

p62 Increases Levels of Proteasome Substrates in a UBA-Dependent Manner

(A and B) Overexpression of full-length, but not truncated, p62 increases levels of soluble polyQ. HeLa cells were transfected with httQ74-HA and either pcDNA (control), pEGFP-p62, or pEGFP-p62ΔUBA at 1:3 ratios and were incubated for 48 hr. (B) Cells were immunoblotted, and bands were quantified by densitometry.

(C) Effect of p62 overexpression on polyQ aggregation is dependent on UBA. SK-N-SH cells were cotransfected with httQ74-HA and either pEGFP-C1 (control), pEGFP-p62, pEGFP-p62ΔUBA, or pEGFP-UBA at 1:3 molar ratios. A total of 24 hr posttransfection, cells were immunostained for HA. The percentage of double-positive cells with httQ74 aggregates was counted.

(D–F) Overexpression of p62 increases levels of p53 and Ub^G76V-GFP. HeLa cells were transfected with Ub^G76V-GFP and either pEGFP-C1 (control), pEGFP-p62, pEGFP-p62ΔUBA, or pEGFP-UBA at 1:3 molar ratios. A total of 24 hr posttransfection, cells were analyzed by immunoblotting. (D) Cells transfected only with Ub^G76V-GFP were incubated with MG132 for 3 hr prior to lysis, as a positive control (D). The asterisk denotes a nonspecific band. (E and F) Levels of endogenous p53 and Ub^G76V-GFP were quantified by densitometry.

For all of the graphs, data are shown as means ± SE for three separate experiments performed in triplicate. ^∗p < 0.05, ^∗∗∗p < 0.005, t test; all other comparisons are not significant (n.s.).