FIGURE 3.

Surfactant protein C (SFTPC) internalisation from the plasma membrane is AP2 dependent and is retarded by the I73T mutation. a) Quantitative SFTPC internalisation assay. Cells expressing green fluorescent protein (GFP)-SFTPC-wild type (WT) or -I73T were labelled with a BRICHOS domain antibody on ice, then protein allowed to internalise at 37°C for the indicated times. Cells were placed back on ice, fixed but not permeabilised, and labelled with a secondary antibody before analysis by flow cytometry. n=3. Data are presented as mean±sem. b) and c) HeLa cells stably expressing GFP-SFTPC-WT or -I73T were treated with 30 µM bafilomycin for 16 h to inhibit clathrin-mediated endocytosis. This results in cell-surface SFTPC accumulation as seen by confocal microscopy and measured by flow cytometry. d) AP2-deficient cells were used to assess localisation of Retention Using Selective Hooks (RUSH)ed GFP-SFTPC-WT and -I73T after 2 h of biotin treatment. AP2M1 CRISPR knockout cells were immunoblotted for the AP2α-subunit. Although some α-subunit remains, this complex is nonfunctional. Scale bars=10 μm.