Fig. 3.

p62 is required for sequestering UB-RFP into punctate structures. (A and B) HeLa cells transfected with either siRNA pools directed against p62 (A) or control siRNA (B). Twenty-four hours later, cells were transfected again with the respective siRNA and also with a plasmid encoding UB-RFP. Four hours after the second transfection, leupeptin (0.25 mM) was added to the cells, and 20 h after that, cells were fixed and stained with anti-p62 and Alexa 543-goat anti-rabbit antibodies. White lines in micrographs illustrating endogenous p62 immunostaining frame the outline of cells coexpressing UB-RFP. Arrowheads in (B) indicate obvious examples of colocalized UB-RFP and endogenous p62. (Scale bars, 10 μm.) (C) Quantification of the percentage of cells with 5 or more punctate structures containing either RFP or UB-RFP and GFP-LC3 in cells transfected with also control siRNA or p62 siRNA and treated with leupeptin as in (A and B). Shown are the averages ± standard deviations from three independent experiments with each experiment including at least 50 cells scored.