Fig. (2).

Transport of Hsp70 to the cell surface involves the endolysosomal system (A) Representative Hsp70 immunoblotting for endolysosomal fractions isolated from “ctrl” and “+Hsp70” cells. See also (Fig. 1S) for characterization of fractionation. (B) Visualization of endosomal localization of Hsp70-mRFP. While releasing a temperature block cells were labeled with transferrin-PacificBlue at 37 °C. Subcellular localization of Hsp70-mRFP is shown after releasing a temperature block (gray image, scale bar= 10 µm). Vesicular structures are displayed in red for Hsp70-mRFP and in green for transferrin-PacificBlue, respectively. A representative overlay image (merge) is shown with a scale bar of 10 µm. (C) Visualization of lysosomal localization of Hsp70-mRFP. Cells were loaded with dextran-CascadeBlue for 2 h and incubated for 16 h at 37 °C. Following a temperature block vesicular structures are displayed in red for Hsp70-mRFP and in green for dextran-CB, respectively. A representative overlay image (merge) is shown with a scale bar of 10 µm. (D) Transport of Hsp70 to the surface in “ctrl” and “+Hsp70” cells in the presence of the specific vacuolar ATPase (endolysosomal system) inhibitor. Cells were incubated with bafilomycin A1 at 37 °C for 30 min prior to measurements. Data are expressed in relation to the values of untreated cells (n ≥ 3; mean ±SD). For absolute values refer to (Fig. 4A). (The color version of the figure is available in the electronic copy of the article).