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. 2022 May 5;221(6):e202105112. doi: 10.1083/jcb.202105112

Figure 7.

Figure 7.

ATG16L1 interacts with V-ATPase during non-canonical autophagy. (a and b) ATG16L1−/− RAW264.7 cells and those re-expressing Flag-tagged WT and K490A ATG16L1, were treated with OPZ for 25 min (a) or STING agonist DMXAA (50 μg/ml) for 1 h (b). Input lysates and Flag immunoprecipitations were probed for ATP6V1A, ATG16L1, and ATG5 by Western blotting. (c) ATG16L1−/− HCT116 cells and those re-expressing FLAG-tagged WT and K490A ATG16L1 were treated with TRPML1 agonist C8 (2 μΜ) for 30 min. Input lysates and FLAG immunoprecipitations were probed by Western blotting as above. (d) ATG16L1−/− RAW264.7 cells and those re-expressing FLAG-tagged WT and K490A ATG16L1 were treated with mTOR inhibitor PP242 (1 μM) for 1 h. Input lysates and Flag immunoprecipitations were probed by Western blotting as above. (e) Confocal images of RAW264.7 cells expressing WT or K490A ATG16L1 treated with OPZ (25 min), DMXAA (1 h), or PP242 (1 h) and stained for LC3. Asterisks denote phagosomes. Scale bar, 10 μm. (f) Confocal images of GFP-rLC3B HCT116 cells expressing WT or K490A ATG16L1 treated with C8 (30 min). Scale bar, 15 μm. (g) ATG16L1−/− HCT116 cells and those re-expressing FLAG-tagged WT and K490A ATG16L1, were treated with BafA1 (100 nM) or SaliP (2.5 μM) for 1 h. Input lysates and FLAG immunoprecipitations were probed by Western blotting as above. Source data are available for this figure: SourceData F7.