JCS037127 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. JAM-A expression in HL60 cells. (A) JAM-A immunoprecipitation of HL60 cells lysed with 1% Triton-X 100 buffer. Immunoprecipitated and total cell extracts were immunoblotted for JAM-A (BV22 mAb). Molecular mass marker bands (kDa) are indicated on the left. (B) FACS analysis of HL60 cells with the BV16 mAb anti-human JAM-A. The thin line corresponds to staining with an irrelevant mAb of an identical isotype.

• Supplemental Figure S2 -

  Fig. S2. JAM-A distribution in activated dHL60 cells. Confocal immunofluorescence analysis of polarized dHL60 (fMLP stimulation for 20 minutes). Two different types of representative cells showing a low or partial polarization are shown. The anti-JAM-A mAb (red) was given to living cells, before paraformaldehyde fixation (see Materials and Methods). The image represents the z-stack projection of about 20-25 confocal sections from the basal to the apical cell side, as indicated by the arrow at the left. The lower panels show the corresponding lateral view of JAM-A intracellular distribution along the cell body. The images represent the maximum projection along the xz axes of confocal stacks of about 15 sections. Scale bar: 5 μm.

• Supplemental Figure S3 -

  Fig. S3. JAM-A distribution in resting dHL60 cells. Confocal immunofluorescence analysis of untreated dHL60 seeded on fibronectin-coated coverslips. As indicated, the anti-JAM-A mAb (red) was given either after paraformaldehyde fixation or to living cells before fixation (see Materials and Methods). Cell nuclei were counter-stained with DAPI (blue). The image represents the z-stack projection of 20-25 confocal sections from the basal to the apical cell side, as indicated by the arrow at the left. Scale bars: 5 μm.

• Supplemental Figure S4 -

  Fig. S4. JAM-A is internalized in intracellular vesicles. The internalization of JAM-A from the plasma membrane was analyzed in dHL60 cells treated with fMLP for 20 minutes. To detect proteins localized at the membrane surface, living cells were treated at 4°C with the antibodies and then fixed and processed for immunofluorescence (a-c). To detect the internalized proteins, cells were treated with the antibodies, acid washed before fixation, and permeabilized with saponin during immunofluorescence analysis (d-f). Images show the merge of double stainings for phalloidin (green) and for mouse IgG (red) (a,d) or VE-cadherin (TEA mAb, isotype IgG1, red) (b,e) or JAM-A (BV16 mAb, isotype IgG1, red) (c,f). The specificity of the assay was confirmed by the undetectable staining obtained using either a pool of mouse IgG (a,d) or a mAb of the same isotype of JAM-A antibody (b,e). JAM-A is poorly detectable at the plasma membrane (c), but is strongly internalized in intracellular vesicles (f). Phalloidin staining observed in cells that have not been treated with saponin is due to the partial permeabilization induced by cell fixation, which allows phalloidin internalization. Scale bar: 5 μm.

• Supplemental Figure S5 -

  Fig. S5. Specific fibronectin-induced clustering of JAM-A. Immunofluorescence staining of dHL60 cells incubated with either fibronectin- or polylysine-coated beads. To verify the specificity of the assay, after incubation with the beads, cells were fixed and stained with antibodies against either JAM-A or another membrane protein, MHC Class I. JAM-A specifically clustered at the contact sites between fibronectin-coated beads and cells, while both fibronectin- and polylysine-coated beads did not induce clustering of MHC Class I. Single projections of z-stacks of about two confocal sections (stack z-spacing, 0.3 μm) are shown. Scale bar: 5 μm.

• Supplemental Figure S6 -

  Fig. S6. Expression of integrin is not modified by JAM-A expression. Western blotting analysis of β1 integrin in neutrophils derived from JAM-A^+/+ and JAM-A^−/− mice. Cells with different levels of activation were analyzed. The upper band represents the immature form of protein. 40 μg of total lysates were loaded. 1, positive control of cells derived from pancreas; 2, JAM-A^+/+ bone marrow cells; 3, JAM-A^−/− bone marrow cells; 4, bone marrow-purified JAM-A^+/+ neutrophils; 5, bone-marrow-purified JAM-A^−/− neutrophils; 6, cells from bone marrow of JAM-A^+/+ mice injected with thioglycolate in the peritoneum; 7, cells from bone marrow of JAM-A^−/− mice injected with thioglycolate in the peritoneum; 8, cells from peritoneal exudates of JAM-A^+/+ mice injected with thioglycolate; 9, cells from peritoneal exudates of JAM-A^−/− mice injected with thioglycolate.