Generation of mice with a megakaryocyte-specific ADAM10 deficiency. (A) The scheme depicts detection of Adam10 wild-type and floxed alleles. The external probe (black horizontal bar) recognizes a sequence upstream of exon 2. Exon 2 is represented as a black vertical bar. The wild-type band between 2 PstI sites is 13.4 kb, whereas the floxed band recognized by the external probe is 9.5 kb. The region used for homologous recombination is indicated. Striped black box: neomycin resistance gene. ▶: lox P site; ♦: FRT site. (B) Southern blot analysis from tail DNA of wild-type (+/+), conditionally targeted heterozygous (+/−), and conditionally targeted homozygous (−/−) mice. − indicates the floxed allele. (C) Western blot of platelet lysates from control and Adam10^{fl/fl, PF4-Cre} mice. Whole platelet proteins were separated by SDS-PAGE and immunoblotted with an anti-ADAM10 antibody. Bands at 60 kDa are the mature protein, whereas bands at 30 kDa show the degraded protein. GPIIIa was used as a loading control. Flow cytometric analysis of platelet count (D) and platelet size (E). (F) Protein expression: platelets were stained with the indicated fluorophore-labeled antibodies for 15 minutes and analyzed directly. (F) Platelet size was determined in a Sysmex cell counter.