Blocking the α4 integrin–paxillin interaction selectively impairs mononuclear leukocyte recruitment to an inflammatory site
J. Clin. Invest. Chloé C. Féral, et al. 116:715 doi:10.1172/JCI26091 [Go to this article.]

Figure 1
Generation of α4(Y991A) mice (A) Representation of targeting vector, WT α4 locus, and the targeted allele before and after CRE recombination (see Methods for details). The tyrosine 991 (star) was mutated into an alanine, along with the creation of a unique restriction site for Eco47III. (B) Southern blot analysis of genomic DNA showing correct targeting of the α4 allele. Genomic DNA was isolated from R1 ES cells, digested with BamHI, and hybridized with the 3′ probe. Probing the same Southern blot with a specific probe for Neo revealed 1 targeted band at the expected size, indicating that there was a single homologous recombination event. (C) PCR analysis of genomic DNA following CRE recombination. Two types of recombination occurred, either between the 2 most external loxP sites (by-product; BP) knocking out both the selection cassette and exon 28, or between the loxP sites flanking the selection cassette (knock-in; KI), selectively removing the cassette. Knock-in clones were subsequently used for blastocyst injection. (D) Southern blot analysis of mouse tail genomic DNA showing correct targeting of the α4 allele. Two lines were obtained. WT, heterozygotes, and homozygotes were confirmed by Southern blot using BamHI-digested DNA and hybridizing with the 5′ external probe. Targeted band at the expected size [α4(Y991A) (YA), 10.6 kb; WT, 14.7 kb] was obtained. (E) Coimmunoprecipitation of α4 integrin with paxillin. Paxillin was immunoprecipitated, and coimmunoprecipitated α4 integrin was visualized by blotting for biotin after separation in SDS-PAGE gel. Immunoprecipitated paxillin was detected by anti-paxillin antibody.