Figure 5.
p62 Mediates USP10-Induced Protein Aggregation
(A) HeLa cells were transfected with p62 small interfering RNA (siRNA) (p62-1 or p62-2) or control siRNA (NT) and were further transfected with the HA-USP10 and the GFP-CFTR-ΔF508 plasmids. The percentages of cells with GFP/HDAC6-positive aggresome are presented as the mean ± SD (n = 4); *p < 0.05; **p < 0.01; ***p < 0.001.
(B) The NP-40-soluble fractions (SF) and NP-40-insoluble fractions (ISF) from the indicated HeLa cells were characterized by western blot (WB) using anti-GFP, anti-Ub, anti-p62, anti-HA, anti-Lamin B, and anti-β-actin antibodies.
(C) HeLa cells were transfected with the p62 plasmid. The cells were stained with the anti-p62 (red) antibody and with anti-Ub (green), anti-HDAC6 (green), or anti-USP10 (green) antibody, and with Hoechst 33258 (blue). The small arrow indicates USP10-negative p62 aggregate, whereas the large arrow indicates USP10-positive p62 aggregate. Scale bars, 10 μm.
(D) HeLa cells were transfected with the p62 and the GFP-LC3 plasmids. The cells were stained with the anti-p62 antibody (red) and Hoechst 33258 (blue). Scale bars, 10 μm.
(E) HeLa cells were transfected with the p62 plasmid and HA-USP10 or its mutant plasmids (USP10C424A, USP101−116, or USP1096−798). The cells were stained with anti-HA (green) and anti-p62 (red) antibodies, and with Hoechst 33258 (blue). Small arrows indicate HA-USP10-negative p62 aggregates, whereas the large arrows indicate HA-USP10-positive p62 aggregates. Scale bars, 10 μm.
(F) The sizes of p62 aggregates (μm2) are presented as the mean ± SD (n = 30); ****p < 0.0001; ND, not detected.
See also Figure S4.