Figure 6.

GRAF1 is a positive regulator of autophagy. (A) HeLa cells transfected with two rounds of control, ARHGAP26, GRAF1a, or GRAF1b siRNA for 5 d were treated with 400 nM bafilomycin A1 (Baf A1) as indicated for 4 h. Cells were lysed and subjected to Western blotting with the indicated antibodies. The data represent the means ± SD of the percentage of LC3-II/actin ratios obtained from three independent experiments. Black lines indicate that intervening lanes have been spliced out. (B) HeLa cells transiently expressing either GFP, GRAF1-GFP, or GRAF1 BAR+PH-GFP for 20 h were treated with 400 nM Baf A1 as indicated for 4 h. Cells were lysed and subjected to Western blotting with the indicated antibodies. (C) HeLa cells transiently expressing either GFP, Arf6-GFP, or Arf6 N48R-GFP for 20 h were treated with 400 nM Baf A1 as indicated for 4 h. Cells were lysed and subjected to Western blotting with the indicated antibodies. (B and C) The data represent the means ± SD of the percentage of LC3-II/actin ratios obtained from two independent experiments. (D) Arf6 may regulate autophagy via multiple pathways: Arf6 activates PIP5K, which leads to the production of PIP₂ and the regulation of endocytosis; Arf6 activates PLD, which leads to the formation of phosphatidic acid (PA), a lipid involved in autophagosome formation. The question whether Arf6 regulates autophagy via GRAF1, a PIP₂-binding protein involved in clathrin-independent endocytosis, remains to be elucidated. Furthermore, Arf6 may regulate autophagy by affecting intracellular membrane flow via additional routes. SE, short exposure; LE, longer exposure.