Fig. 6: In vitro cleavage of ATG16L1 by active recombinant caspase-6.

a HA-tagged ATG16L1 were overexpressed in HEK 293T cells and immunoprecipitated with anti-HA antibody. Immunoprecipitants were incubated 16 h at 37 °C with active recombinant caspase-6 in absence or presence of inhibitor Z-VEID-fmk (50 μM) and ATG16L1 cleavage was assessed by western blot with anti-HA and anti-ATG16L1 antibody. b THP-1 macrophages were immunoprecipitated with anti-ATG16L1 antibody and immunoprecipitants were incubated 16 h at 37 °C with active recombinant caspase-6 in absence or presence of inhibitor Z-VEID-fmk (50 μM) and ATG16L1 cleavage was assessed by western blot with anti-ATG16L1 antibody. Direct cell lysate was used as a control (lane 5). c HA-tagged ATG16L1 overexpressed HEK 293T cells were incubated with Eh for 10 min in absence or presence of capspase-6 (Z-VEID-fmk), pan-caspase (Z-ZVAD-fmk), caspase-3 (Z-DEVD-fmk), caspase-8 (Z-IETD-fmk) inhibitors (50 μM) and ATG16L1 cleavage was assessed by western blot with anti-ATG16L1 antibody. d MCF-7 cells (caspase-3 defective cell line) were incubated with Eh for 20 min and with Eh for 30 min in absence or presence of capspase-6 (Z-VEID-fmk), pan-caspase (Z-ZVAD-fmk), caspase-8 (Z-IETD-fmk) inhibitor (50 μM) and ATG16L1 cleavage was assessed by western blot with anti-ATG16L1 antibody. Cells without treatment is symbolized as (Ctrl). Eh-macrophage 1:20 ratio used for (c, d). e N-terminal GST-tagged recombinant ATG16L1 incubated with recombinant active caspase-6 for 16 h at 37 °C and ATG16L1 degraded fragments were assessed by western blot with anti-ATG16L1 (M150-3, MBL international) and anti-GST antibody. Data are representative of at least three independent experiments (n = 3). f Sequence alignment of amino acid calls from Edman degradation analysis of 70 kDa band and schematic presentation of the fragment with a cutting site at aspartic acid 495 (D495) position. Blue line indicates GST protein (M1-K218) and black line for ATG16L1 (M85- D495).