Quantification of the amount of CD9P-1 associated with tetraspanins.

<p>The amount of CD9P-1 associated with tetraspanins or that remaining outside the tetraspanin complexes was quantified in HEK-293 or HEK/CD9P-1 cells after transfection with CD9P-1 or/and CD9. Cells were lysed using the mild detergent Brij97. CD9P-1 associated with tetraspanins was investigated by depletion of tetraspanins from the lysate. Three rounds of subsequent immunoprecipitation were performed with mAbs directed against CD9 and CD81. After depletion of tetraspanin complexes, the amount of CD9P-1 still remaining in the lysate and therefore not associated with tetraspanins was measured by three additional rounds of immunoprecipitation using a mAb directed against CD9P-1 (1F11) antibody. All immunoprecipitates were separated by electrophoresis under reducing conditions and proteins were stained using colloidal Coomassie blue. Gels were scanned using the Odyssey device (upper panel). Signals obtained from CD9P-1 were measured and quantitated. When HEK-293 cells were transiently transfected with CD9P-1 (mfi = 300 <i>versus</i> 50 from endogenous), more than 70% of CD9P-1 was associated with tetraspanin partners CD9 (mfi = 150) and CD81 (mfi = 300). Furthermore, HEK-293 cells were transfected with both plasmids encoding CD9P-1 and CD9. Under these conditions when CD9 expression level was higher (mfi = 700), we observed that 100% of CD9P-1 was associated with tetraspanins. In HEK/CD9P-1 cells that stably expressed CD9P-1 with a high level (mfi = 1000), the amount of CD9P-1 associated with tetraspanins was estimated to be 30%. When HEK/CD9P-1 cells were transfected with CD9 (mfi = 700), a higher amount, corresponding to 50% of CD9P-1, was associated with tetraspanins CD9 and CD81 (lower panel). A representative experiment is shown. IP: immunoprecipitation; Tr: transfection.