Figure 1.

Cells expressing Atg4B^C74A exhibit a block in autophagic degradation. (A) MCF7 cells stably expressing GFP-LC3 were infected with adenovirus bearing mStrawberry (Mock), mStrawberry-Atg4B^WT (WT), or mStrawberry-Atg4B^C74A (C74A) and incubated for 40 h. The cells were then cultured in HBSS for 2 h, fixed, and observed using fluorescence microscopy. Bar, 10 μm. (B) PC12 cells were infected with adenovirus bearing GFP (−), 3xFlag-tagged wild-type Atg4B (WT), Atg4B^C74A (CA), or Atg4B^C74S (CS). Cells were cultured either in growth or starvation medium, and lysates were examined by Western blotting using each antibody. Top panel, anti-Flag; middle panel, anti-LC3; bottom panel, anti-α-tubulin. (C) 293A cells stably expressing empty vector (Mock) or mStrawberry-Atg4B^C74A (Atg4B^C74A) were grown in growth medium (F), HBSS (S), or HBSS with 100 nM wortmannin (W) for 2 h. Cell lysates were examined by Western blotting using each antibody. From top panel, anti-RFP, anti-p62, anti-LC3, and anti-α-tubulin. (D) 293A cells stably expressing empty vector (Mock) or shRNA against LC3 (LC3 KD) were cultured in HBSS for 2 h and collected. Cell lysates were examined by Western blotting using each antibody. From top panel, anti-LC3, anti-GABARAP, and anti-α-tubulin. (E) Mock, mStrawberry-Atg4B^C74A-expressing (Atg4B^C74A), or LC3-knockdown (LC3-KD) 293A cells were grown in growth medium (F), HBSS (S), or HBSS with 100 nM wortmannin (W) for 2 h. Long-lived protein degradation was scored as described in Materials and Methods.