Fig. 4

SpHtp3 is taken up via a gp96-like receptor. a Uptake inhibition of SpHtp3-mRFP into RTG-2 cells pre-incubated for 1 h with the inhibitors dynasore, brefeldin A or nystatin (top) and respective quantification (bottom). Nuclei are indicated by dashed lines. Error bars denote s.e.m. (cells: 50). ***p < 0.001, **p < 0.01 (one way ANOVA). Scale bar: 20 µm (n = 3). b FACS analysis of the saturable uptake of SpHtp3-mRFP into RTG-2 cells at higher concentrations. Error bars denote s.e.m. (n = 3). c Appearance of an additional shifted spot (arrowhead) in 2D-PAGE of a RTG-2 cell lysate for a gp96-like protein after incubation with SpHtp3 compared to control samples (c) at different pH values. d Elution fraction of a pull down experiment of an RTG-2 lysate with SpHtp3. An additional band (arrowhead) appears in the pull down with SpHtp3-mRFP, compared to a control with lysate only. LC-MS/MS analysis identified peptides for a gp96-like protein. e In vitro complex formation of recombinant SpHtp3-His₆ with recombinant gp96 (from dog) after cross-link verified by detection by an anti-His antibody. An additional band which only appears in the sample with both proteins is highlighted (complex) (n = 3). f Knock down of gp96 in A549 cells by siRNA by 50%, left: western blot of control cells and the siRNA-treated sample, right: quantification of bands from the western blot. Error bars denote s.e.m. (n = 2). g Reduction of gp96 protein results in a reduced uptake of SpHtp3 into A549 cells with fewer vesicles per cell (see magnifications). Error bars denote s.e.m. (cells: 50). ***p < 0.0001 (t-test). Scale bar: 20 µm (n = 2). h Cell surface exposure of gp96 in human A549 cells at different pH as indicated. 2.5 μg α-gp96 antibody was incubated with living cells to exclude intracellular gp96 and to stain surface exposed gp96 only. Pictures were taken with a Zeiss Imager M2. Scale bar: 20 µm. Graph represents the fluorescence intensity of one cell for each pH as indicated by dashed lines (n = 2)