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. 2011 Jan 10;192(1):17–27. doi: 10.1083/jcb.201009067

Figure 4.

Figure 4.

The PB1 domain but not LRS of p62 is essential for localization to the autophagosome formation site. (A and B) p62 KO MEFs stably expressing GFP-p62 wild type, GFP-p62 LRS mutant 1 (L343A), GFP-p62 LRS mutant 2 (D337A, D338A, and D339A), GFP-p62ΔC (1–265 amino acids), and PB1 mutant (K7A and D69A) were cultured in starvation medium for 1 h. Cells were analyzed by immunofluorescence microscopy using anti-LC3 antibodies. GFP-p62 positivity (%) of the LC3 puncta is shown in B. Data represent mean ± SEM of 30 images. (C and D) p62 KO MEFs stably coexpressing HA-ULK1 and one of the GFP-p62 described in A were cultured in starvation medium containing 0.2 µM wortmannin for 1 h. Cells were analyzed by immunofluorescence microscopy using anti-HA antibodies. GFP-p62 positivity (%) of the LC3 puncta is shown in D. Data represent mean ± SEM of 30 images. (E) p62 KO MEFs stably expressing the indicated GFP-p62 were cultured in regular or starvation medium for 1.5 and 3 h. Cell lysates were analyzed by immunoblot analysis with the anti-p62 and β-actin antibodies. Signal color is indicated by color of typeface. St. M., starvation medium; WM, wortmannin; WT, wild type. Bars: (white) 10 µm; (yellow) 1 µm.