Figure 6. Oligomerization of p62 promotes recruitment of p62 and LC3B to ubiquitin-coated beads in HeLa cells. .
(A) Anti-LC3B immunofluorescence analysis of HeLa cells transiently transfected with mCherry-p62 variants. Nuclei were stained with DAPI. Insets show magnifications of the indicated squares. Scale bars, 5 µm. (B–G) Quantification of mCherry-p62 and GFP-LC3B recruitment around artificial cargo particles in HeLa cells. (B) Schematic outline of the experiment. (C) Western blot analysis of HeLa cell lysates overexpressing wild–type mCherry-p62 with or without silent mutations in the siRNA targeting region. (D) HeLa cell co-expressing siRNA resistant wild-type mCherry-p62 and GFP-LC3B. Endogenous p62 was silenced by siRNA (see Figure 6—figure supplement 1). The arrows indicate co-localization of mCherry-p62 and GFP-LC3B at a BFP-ubiquitin-coated 2 µm bead. Scale bar: 5 µm. (E) Quantification of mCherry-p62 variants localizing to BFP-ubiquitin-coated beads in mCherry-p62 and GFP-LC3B co-expressing cells. (F) Quantification of co-localization of mCherry-p62 variants and GFP-LC3B at BFP-ubiquitin-coated beads. (G) Quantification of GFP-LC3B localization to BFP-ubiquitin-coated beads. For all data in (D–G), averages and SD of three independent replicates are shown. Indicated p-values were calculated by a two-tailed equal-variance Student’s t-test. All graphs show the averages and SD. (E–G) Total beads quantified per condition: wild-type = 113 beads, K7A/D69A = 145 beads, delta PB1 = 117 beads, NBR1-p62 chimera = 120 beads, mCherry = 144 beads.
Figure 6—figure supplement 1. (A) Representative picture of 2 µm latex beads cross-linked with BFP-ubiquitin.
