Involvement of Abi1 in megakaryocytopoiesis and spreading. (A) Colocalization of WAVE2 and Abi1 in a fibrinogen-adherent ES cell–derived MK in the presence of 100 nM PMA (arrows). Right graph shows the intensities of WAVE2 expression (red) or Abi1 expression (green) between “a” and “b” by NIH J-Image. (B) Immunoblots of Abi1, WAVE1, WAVE2, and tubulin from lysates during early megakaryocytopoiesis. (C) Flow cytometric dot plots of side and forward scatter profiles of 10 000 viable cells staining with anti-GPIbα (PE) antibody on day 12. Large MKs transduced with Abi1 shRNA appeared fewer than the number of large control MKs. (D,E) On day 12, ES cell–derived MKs were plated on fibrinogen for 60 minutes in the absence (D) or presence (E) of 100 nM PMA and were analyzed as depicted. (D) GPIbα expression by MKs in which Abi1 shRNA or scrambled control shRNA was transduced. (E) Arrows indicate peripheral normal lamellipodia. Arrowheads indicate filopodia-rich regions. GFP⁺ or GFP⁻ cells transduced with control shRNA or Abi1 shRNA were assessed for lamellipodia-dominant morphology in the presence of PMA. A total of 3 independent experiments were performed. See “Image acquisition for platelet and MK spreading” for complete image acquisition information.