Fig. 3

Ate1 assembles into p62 bodies. Ate1 KO^Ate1-1 cells were incubated with 10 μM MG132 and/or 20 μM CQ for 6 h (a, b) or 12 h (c, d). a Immunostaining was performed using antibodies against Ate1 (green), p62 (red), and poly-Ub (pUb, magenta) and visualized with confocal microscopy. Insets show a higher magnification of puncta where Ate1 and p62 are colocalized (white arrows). b Quantification of the size (left panel) and number (right panel) of p62 bodies in Ate1 KO^Ate1-1 cells. CONTROL, n=50/6; MG132, n=176/6; STV/CQ, n=138/5; STV/CQ+MG132, n=16/5 bodies/cells were analyzed. The relative area covered by and the number of p62-labeled inclusion bodies were obtained from maximum intensity projections of confocal z stacks. c Immunoblot of Ate1-1, p62 and poly-Ub proteins levels in reducing conditions. d Total homogenates of treated cells were separated by SDS-PAGE without β-mercaptoethanol (nonreducing) and western blotted. p62 was used as an endogenous positive control. Oligo.: oligomers, Mono.: monomer. (c, d) Ate1-1 was detected using an antibody against Ate1, with Gapdh as a loading control. Statistical significance was assessed using a two-sided unpaired t test. *P≤0.05, ***P≤0.001, ns=nonsignificant. Scale bars: 20 μm (main images), 3 μm (inset)