Figure 1.

Generation of megakaryocyte lineage-restricted Dab2 knockout mice. A, Dab2 targeting strategy (left panel). The positions of the PF4-Cre (450 bp) and the wild-type (460 bp, +), null (250 bp, −) and floxed (530 bp, fl) alleles for DAB2 were indicated (right panel). B, The PCR product was sequenced and truncated DAB2 transcript was found to present in the −/− megakaryocyte. C, The lysates of the sorted megakaryocytes were collected to analyze Dab2 expression by Western blot using anti-Dab2 antibody. Cell lysates from the small cell fraction on the top of BSA gradient were also collected for the use as non-megakaryocytic small cells (NC) to demonstrate lineage-specific knockout of Dab2 protein. D, Dab2 expression in the fl/fl and −/− megakaryocytes was analyzed by immunofluorescence staining using anti-Dab2 antibody followed by Alexa Fluor 546 goat anti-rabbit secondary antibody (red) and counterstained with 4′,6-diamidino-2-phenylindole (DAPI, blue). The images were observed using fluorescence and differential interference contrast microscopy. Left panel, the upper images represent the fluorescent field of XY projection and the bottom images represent the fluorescent XY projection merged with its corresponding bright-field image (length of bar = 20 μm). The fluorescence intensity of fl/fl (n = 40) and −/− (n = 40) megakaryocytes was quantified by ImageJ software and was shown as Box and Whisker plot (right panel). The ends of the box are the upper and lower quartiles and the median is marked by a horizontal line inside the box. The whiskers are the two lines outside the box that extend to the highest and lowest values. **, p < 0.01. E, The lysates of fl/fl and −/− platelets were analyzed by Western blot using anti-Dab2 antibody. The position of membrane corresponding to the molecular weight of 82 kDa was marked. The expression of β-actin was used for the control of equal protein loading.