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Fig. 4. Co-clustering of {alpha}6ß4 with D6.1A. (A) Progressor cells were incubated for 15 minutes at 37°C with A2.6, C4.4, D6.1, D5.7 (all of which recognize metastasis-associated molecules), anti-CD9 (expressed on Progressor cells) and, as a negative control, anti-CD18 (not expressed on Progressor cells). Cells were washed with cold PBS and were incubated for 20 minutes at 37°C with an excess of FITC-labeled anti-mIgG. Thereafter cells were immediately transferred on ice. After washing and fixation, free binding sites of FITC-labeled anti-mIgG were blocked by incubation with PBS containing 10 µg/ml mouse IgG. Finally, cells were stained with B5.5-TxR (1 hour, 4°C), the staining solution also containing mIgG to avoid unspecific binding of B5.5-TxR to the FITC-labeled anti-mIgG. (B) Progressor cells were mixed with BSp73AS-D6.1A (indicated by one asterisk) that express the D6.1A tetraspanin, or BSp73AS-ß4 cells (indicated by two asterisks) that express {alpha}6ß4. As described in A, cells were stained with D6.1/anti-mIgG-FITC and B5.5-TxR or with B5.5/anti-mIgG-FITC and D6.1-TxR. Single stainings and overlays of green and red fluorescence are shown. The FITC-labeled anti-mIgG does not bind unspecifically (anti-CD18 staining) and B5.5-TxR or D6.1-TxR does not bind to FITC-labeled anti-mIgG that was blocked by mIgG (staining of the mixture of Progressor and BSp73AS-D6.1A or BSp73AS-ß4 cells). Although Progressor cells express CD44v6, C4.4A, D6.1A and D5.7A at a comparably high level, {alpha}6ß4 co-clustered only with CD44v6 (A2.6) and D6.1A. Scale bar: (A) 10 µm; (B) 5 µm.