Figure 3.

LC3B Competes with FIP200 for p62 Binding, and FIP200 Is Excluded from the Autophagosomal Lumen

(A) RFP trap beads were coated with mCherry-p62 FIR 4P and incubated with the GFP-FIP200 CTR (aa 1458-1594, 1.1 μM). Then, increasing concentrations of LC3B were added. Beads were imaged by microscopy. The intensities of the GFP and mCherry signals on the beads were measured and plotted against the LC3B concentrations (lower panels). A zoom-in of the plot at the lowest LC3B concentrations is shown on the left. Average intensities and SD for n = 3 are shown. Negative and positive controls of binding are shown in Figure S3B.

(B) HeLa cells were left untreated, starved or treated with puromycin both in presence or absence of bafilomycin. Cell lysates were then treated with proteinase K in the presence or absence of Triton X-100 and analyzed by western blotting with anti-p62 and anti-STX17. LC3B processing was used to monitor autophagy induction. The percentage of protease protection for FIP200, in comparison to p62 and STX17 is plotted on the right. Average protection and SD for n = 3 are shown. Protease protection in starved cells treated with wortmannin and/or bafilomycin are shown in Figure S3D.

See also Figure S3.