Fig 4.

p62 delivers ubiquitin and microbicidal ubiquitin-derived peptides to the mycobacterial phagosomes. (A - B), Autophagy promotes ubiquitin colocalization with mycobacterial phagosomes. RAW264.7 cells were infected with Alexa-568 labeled BCG and chased for 1 h in complete media. Cells were then subjected to autophagic induction for 4 h by starvation. Cells were fixed and stained for ubiquitin conjugates. (C - D) p62 is necessary for the autophagic targeting of ubiquitin conjugates to the mycobacterial phagosomes. p62 was depleted from RAW264.7 cells with siRNAs. At 48 h after transfection, cells were infected with Alexa-568 labeled BCG, subjected to autophagic induction, and stained for ubiquitin conjugates as in (A and B). Data, means ± SEM from at least three independent experiments. At least 50 phagosomes per category per independent experiment were quantified using % BCG-marker colocalization; **, p < 0.01, *, p < 0.05, all relative to control. For Pearson's colocalization coefficient (defined in Experimental Procedures) exact p values are shown and number of phagosomes analyzed in each category indicated. See also Fig. S5.