Fig. 6. CD41 and CD61 are associated in EB6 cells. (A) Western blot analysis was performed in reducing conditions with 30 µg of protein from unfractionated EB6 (U), CD41+ (+) and CD41– (–) cells obtained after immunomagnetic purification and 5 µg of proteins from murine platelets (Pts) used as a control. The anti-CD41 MoAb (anti-GPIIb) (the same MoAb used for purification and cell sorting) detected a doublet at 125 kDa. This doublet was at the threshold of detection in the CD41– cell fraction and unfractionated EB6 cells. These data were confirmed with a polyclonal antibody directed against human CD41, which crossreacts with the murine protein. CD61 (GPIIIa) was detected as a 90 kDa protein, with a rabbit anti-CD61 human antibody used to probe the CD41+ cell fraction and mouse platelets. This band was less intense in the CD41– cell fraction and in the unfractionated EB6 cells. The expression of GPIIb and GPIIIa was drastically increased in the CD41+ versus CD41– cell fractions, when results are normalized to actin expression. (B) The anti-CD41 MoAb was used to immunoprecipitate CD41 protein from unfractionated EB6 cells (EB6) (300 µg of protein) and murine platelets (Pts) (30 µg of protein). Bone marrow cells from CD41 knockout mice (BM) and Baf3 cells (Baf3) (300 µg of proteins), were used as a negative control in these experiments. The immunoprecipitate was probed with rabbit anti-human CD41 and anti-human CD61 antibodies. Two major bands were detected: one at 125 kDa, corresponding to CD41, and another at 90 kDa, corresponding to CD61 in EB6 and platelets (but not in the negative controls). These bands were not detected when the immunoprecipitate was probed with a pre-immune rabbit serum (data not shown).
