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. 2019 Apr 1;38(9):e100554. doi: 10.15252/embj.2018100554

Figure 1. ATG16L1 membrane targeting activity is retained in the absence of ATG5 or WIPI2.

Figure 1

  1. Analyses of protein expression in lysates of various cell lines by Western blotting against the indicated antibodies.
  2. Fluorescence analyses of GFP‐ATG5 or ATG16L1‐GFP stably expressed in ATG5−/− or ATG16L1−/−. Cells were amino acid starved for 2 h prior to fixation and imaging of the GFP fluorescence. Scale bar: 10 μm.
  3. Assessment of ATG16L1 levels in the cytosolic (C) and membrane (M) fractions in lysates of the indicated cell lines using Western blot analyses and antibodies against ATG16L1. ATG16L1−/− and ATG5−/− stably expressed ATG16L1‐GFP while ATG3−/− stably expressed Flag‐S‐ATG16L1. Antibodies against α‐tubulin and integrin β1 were used as controls for fractionation. Exogenous (exo) ATG16L1 was detected using antibodies against ATG16L1.
  4. Lack of WIPI2 expression is confirmed by Western blot analyses in wild‐type MEFs (WIPI2+/+) and WIPI2−/− cells.
  5. Immunofluorescence analyses of ATG16L1−/− and WIPI2−/− stably expressing Flag‐S‐ATG16L1. Cells were amino acid starved (AA starve) for 2 h in the presence or absence of 3′MA (and additional pretreatment for 30 min) followed by fixation and immunostaining using antibodies against Flag tag to detect ATG16L1. Scale bar: 9 μm. Right panel represents quantification of three independent experiments and error bars depicting SEM values. *P ≤ 0.05, **P ≤ 0.01 (pairwise unpaired Student's t‐test).