p62 is colocalized with GM-CSF–induced LC3⁺ puncta and degraded by autophagy in activated monocytes. (A) Human primary monocytes were incubated with or without GM-CSF for 14 hours, and then treated with DQ-BSA for another 2 hours. Cells were fixed and stained with anti-LC3 Ab, followed by an FITC goat anti–mouse IgG (green). Representative confocal images of colocalization of LC3 and red fluorescence of DQ-BSA are shown. (B) Human primary monocytes were pretreated with cycloheximide (CHX) at 1μM or CQ at 50mM, and then incubated with GM-CSF (10nM) for the indicated times and analyzed by immunoblotting against p62. One representative experiment from at least 3 independent experiments is shown. Asterisk indicates one special band of truncated p62 Ab. (C) Human primary monocytes were incubated with or without GM-CSF for 14 hours, and then fixed and costained with anti-p62 Ab followed by a FITC goat anti–mouse IgG (green) and anti-LC3 Ab and then rhodamine goat anti–rabbit IgG (red). Representative confocal images of colocalization of p62 and LC3 are shown. (D) Human primary monocytes were incubated with or without GM-CSF for 14 hours and DQ-BSA for another 2 hours, then fixed and stained with anti-p62 Ab, followed by an FITC goat anti–mouse IgG (green). Representative confocal images of colocalization of p62 and red fluorescence of DQ-BSA are shown. Scale bars indicate 5 μm. Arrows indicate colocalization of green and red fluorescence (A,C,D).