Generation of platelet-specific sGC β₁ deficient mice. (A) Targeted disruption of sGC β₁ gene. Top: structure of sGC β₁ gene. Top middle: sGC β₁ gene targeting vector, containing a pMC-Neo gene that is flanked with sGC β₁ genomic sequence, and PGK-TK that is cloned 3′ to the sGC β₁ sequence. Bottom middle: the mutated locus showing the site of PCR used for genotyping. Exons 7 and 8 of sGC were flanked with LoxP sites. Bottom: pMC-Neo gene and exons 7 and 8 were removed by crossbreeding of the sGC conditional mice with specific Cre transgenic mice. (B) PCR genotyping of DNA from offsprings of sGC LoxP chimeras. PCR amplifies a 362-bp fragment from the wild-type allele and a 402-bp fragment from heterozygotes carrying a loxP site (sGC^fl/fl) in the sGC β₁ allele. (C) Detection of sGC β₁ subunit in mouse platelets by Western blot. Washed platelets from C57BL/6J, sGC β₁^fl/fl, and sGC β₁^fl/fl/Pf4-Cre mice were solublized in 1 × SDS sample buffer. sGC β₁ in platelet lysates was detected by Western blot with a polyclonal antibody against sGC β₁. β-actin was used as a loading control. (D) Washed platelets were incubated with selected concentrations of SNP at 37°C for 10 minutes. The reaction was stopped by adding an equal volume of ice-cold 12% (wt/vol) trichloroacetic acid, and cGMP concentrations were determined using a cGMP enzyme immunoassay kit. Means and standard deviations of triplicates are shown from one representative experiment of 3. (E) Washed platelets were incubated with SNAP1 (100μM), SNP (100μM), or forskolin (10μM) at 37°C for 10 minutes. VASP phosphorylation was analyzed by Western blot with monoclonal antibodies specifically recognizing phosphorylated VASP at residues Ser²³⁹ or Ser¹⁵⁷. Data shown are representative of 3 independent experiments.