Fig. 3

Internalization and subcellular localization of cargo antibodies by sorting and cell surface receptors. A Representative images (maximum projected z-stacks) showing immunofluorescence total staining of EEA1 (magenta) and 24 h cargo antibody against sortilin (green) in hiPSC-BECs with IMARIS spot analysis of the fluorescent signals. Scale bars are 10 µm. The white square inset is magnified in micrographs ij-il with scale bars showing 2 µm. B, C Colocalization analysis on hiPSC-BECs (B) and Caco-2 cells (C) of internalized cargo antibodies with selected markers in relation to the steady-state receptor (total receptor in black bars). See materials and methods for calculation of the colocalization score. See also Additional file 2: Fig S3 for the total spot measurements of receptor and cargo antibodies. D, E As an approximation for intracellular cargo antibody accumulation, the cargo antibodies colocalizing with the four intracellular markers were summed and analysed in relation to TfR cargo antibody accumulation. D Bar plots with sums for hiPSC-BECs and (E) for Caco-2 cells. Statistical significance was determined using two-way ANOVA with Tukey´s multiple comparisons test from three independent experiments. See also Additional file 2: Figs S3, S4