Figure 4. Physiological role of the interaction between ALFY and GABARAP.

A HeLa FlpIn GFP-GABARAP cells were treated with proteasomal inhibitor (MG132, 2 h) or subjected to amino acid starvation (EBSS, 2 h), before staining with anti-ALFY antibodies. Scale bar, 10 μm.

B GFP-tagged full-length ALFY wild-type or LC3-interacting region (LIR)-mutant was expressed into Alfy-deficient MEFs using an adenovirus system. At 48 h after infection, the MEFs were cultured in normal media or EBSS for 1.5 h before staining with anti-LC3B or anti-GABARAP antibodies. Scale bars 10 μm.

C HeLa cells were treated with control or siRNA targeting GABARAP, GABARAPL1 and GABARAPL2. 72 h after transfection cells were immunostained with anti-LC3B, anti-ALFY and anti-p62 antibodies. Scale bars, 10 μm.

D HeLa cells were treated with control or siRNA targeting ALFY. 72 h after transfection, cells were treated with puromycin with or without bafilomycin A1 for 2 h and total cell lysates were fractionated into TX-100-soluble and insoluble fractions. The TX-100-soluble/insoluble fractions were then immunoblotted with the indicated antibodies. Data are representative of three independent experiments.

E ALFY WT and KO MEFs were treated with puromycin or EBSS with or without bafilomycin A1 for 2 h and the total cell lysates were then fractionated into Triton X-100 (TX-100)-soluble and insoluble fractions. The TX-100-soluble/insoluble fractions were then immunoblotted with the indicated antibodies. Data are representative of three independent experiments.

F Schematic model of ALFY-mediated selective autophagy.

Source data are available online for this figure.