JCS01288 Supplemental Figures

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1.The ability of macrophages that are osmotically pulsed (as in main text Fig. 6A) to form Y-Ae⁺ complexes from exogenously delivered GST-Ea protein and to endocytose Cy3-labelled maleylated-BSA, appears unimpaired when compared with untreated cells (main text Fig. 6B). FITC labeled 10 kD dextran (F-Dex) in the cytosol was used to visualize the pulsed cells using osmotic lysis of pinosomes.

• Supplemental Figure 2 -

  Fig. S2. BMC-2 cells that were incubated with 50 mg/ml FITC-streptavidin protein in the fluid phase (top), delivered into the cytosol via coupling to the Chariot™ reagent (middle), or were delivered via osmotic pinosome lysis (bottom) were washed, pulse-chased for 15 minutes at 37°C, fixed and imaged on a wide-field microscope equipped with fluorescein optics. Note that the Chariot reagent introduces the FITC-streptavidin into the cytosol as effectively as the osmotic lysis, whereas the fluid-phase pulse remains confined to punctate endosomal structures.

• Supplemental Figure 3 -

  Fig. S3. Y-Ae⁺ peptide MHC class II complexes in macrophage APCs from B10A.(5R) mice do not colocalise with antibodies directed against the (A) invariant chain (In-1), (B) ER resident protein ERp57 or (C) trans-Golgi protein TGN-38. Scale bar, 10 mm.

• Supplemental Figure 4 -

  Fig. S4. Macrophage APCs from C57/Bl6 (A, C) or H-2Ma (–/–; B, D) mice were loaded cytosolically with GST-Ea_52-68-myc, stained with Y-Ae (A, B) or anti-CLIP-MHC II-specific 15G4 (C, D), together with Y3P (anti-I-A^b) antibodies, followed by imaging on a wide-field microscope (A, B) or confocal microscope (C, D). Scale bar, 10 mm.