a) Time-course analysis of p62 distribution in Triton X-100 soluble (top) and insoluble fractions (middle), as well as total p62 protein (bottom) in HEK293T cells after MG132 treatment, as indicated. Actin served as loading control. Note the gradual decrease of soluble p62 with concomitant increase of insoluble p62, as well as the marked increase of total p62 at hour 12. n = 3 independent experiments. ( b and c) MG132-induced turnover of soluble p62 is autophagy-dependent. ( b) Beclin 1 knockdown in HEK293T cells suppressed MG132-induced depletion of Triton X-100-soluble p62 (compare lanes 3 with 2). 5 nM siRNA against beclin 1 was effective in reducing the expression of Beclin 1 protein (middle panels). ( c) Bar graph representation of the soluble p62 levels, which were quantified and normalized against loading control Actin, as indicated. Scale bars indicate s.e.m. n = 9 dishes for each condition pooled from 3 independent experiments ∗ p < 0.05 using analysis of variance + Bonferroni test. ( d) Htt does not affect p62 level under basal and starvation conditions in HEK293T cells. Under both conditions, si-Htt treated samples showed similar levels of p62 protein as in controls of mock- or control siRNA-treated samples. Note the overall reduced level of p62 protein under starvation condition (EBSS incubation). n = 3 independent experiments. ( e) Htt does not affect p62 self-polymerization. in vitro co-IP assay to assess the interaction between HA- and Myc-tagged p62 in transfected HEK293T cells. Similar levels of Myc-p62 were pulled down by HA-p62 between the siHtt-treated (lanes 3 and 4) and control siRNA-treated (lane 2) samples. n = 3 independent experiments. ( f– i) Htt specifically affects the binding affinity between endogenous p62 protein and substrates with Ub-K63, but not Ub-K48, modifications. Co-IP experiments against endogenous ubiquitin using ( f) anti-Ub-K63 or ( g) anti-Ub-K48 from HEK293T cells, or using anti-HA antibody from HEK293T cells transfected with HA-tagged ( h) Ub-K63 (HA-Ub-K63) or ( i) Ub-K48 (HA-Ub-K48). In both cells, Htt knockdown significantly reduced the amount of endogenous p62 that co-immunoprecipitated with Ub-K63 modified proteins, but showed no obvious effect on that co-immunoprecipitated with Ub-K48 modified proteins (compare lanes 2 and 3 with lane 1 in both ( f) and in ( g), and in both ( h) and in ( i)). n = 3 independent experiments.