Subcellular localization of endogenous GABARAPL1 and overexpressed Stbd1 with Atg8 family interacting motif (AIM) mutations in COS M9 cells. Overexpressed full length or mutated hStbd1 with a C-terminal HA-tag and endogenous GABARAPL1 in COS M9 cells was immunostained with anti-HA antibodies (red) or anti-GABARAPL1 antibodies (green). (A) Co-localization of hStbd1 and GABARAPL1 (merged in left panel) in cells expressing C-terminal HA-tagged full length hStbd1 (right panel) and endogenous GABARAPL1 (middle panel). (B) Loss of co-localization (merged in left panel) of endogenous GABARAPL1 (middle panel) with potential AIM deletion mutant of hStbd1, Δ198–222–HA (right panel). (C) Impaired co-localization (merged in left panel) of endogenous GABARAPL1 (middle panel) with double mutation in potential AIM on hStbd1, (W203A, V206A)-HA (right panel). (D) Unaffected co-localization (merged in left panel) of endogenous GABARAPL1 (middle panel) with double mutation in another potential AIM on hStbd1, (W212A, V215A)-HA (right panel). Nuclei were stained with Hoechst (blue). The scale bar is 20 µm.