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Fig. 7. L6-Ag is associated with tetraspanins. (A) HT1080 cells were surface labelled with EZ-link Sulfo-NHS-LC-biotin and lysed in 0.8% Brij98/0.2% Triton X-100. Protein complexes were immunoprecipitated with anti-L6-Ag mAb (lane 1), anti-CD81 and mAb M38 (lane 2) or control mAb (lane 3). Immunocomplexes were separated in 11% SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were developed with streptavidin conjugated to horse radish peroxidase (HRPO). (B) HT1080 cells were lysed in either 0.8% Brij98/0.2% Triton X-100 or 0.5% CHAPS. Protein complexes were immunoprecipitated with anti-L6-Ag mAb (lane 2), anti-CD151 mAb 5C11 (lane 3), anti-CD81 mAb M38 (lane 4) or anti-CD63 mAb 6H1 (lane 5). Irrelevant mAb (187.1) was used as a negative control (lane 6). The protein lysate (lane 1) was used as a positive control. Immunocomplexes were separated in 11% SDS-PAGE and transferred onto nitrocellulose membranes. Membranes were developed with the mAbs to L6-Ag (L6pke) and with polyclonal anti-CD151 Ab. (C) Co-localisation of L6-Ag with tetraspanins on the surface of BT549 cells. Cells grown on glass coverslips were fixed with 2% paraformaldehyde and subsequently stained with combinations of anti-L6-Ag plus anti-CD63 mAbs (or anti-L6-Ag plus anti-CD9 mAb). Staining was visualised using Alexa-Fluor-594-conjugated goat anti-mouse IgG2a Ab (red) and Alexa-Fluor-488-conjugated goat anti-mouse IgG1 Ab (green). Scale bar: 15 µm.
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