Epb4.2 gene targeting results in a null mutation. (a) Restriction map of the Epb4.2 locus with the targeted allele shown below. Numbered boxes represent exons. Upon homologous recombination in ES cells, a neomycin-resistant cassette (hatched box) replaced a segment of the gene extending from intron 3 to exon 8. A 667-bp 5′-flanking KpnI-NsiI fragment (filled bar) was used as the hybridization probe to distinguish alleles by Southern blot analysis. Kp, KpnI. Ns, NsiI. Xb, XbaI. Ec, EcoRI. Inset: Southern blot analysis. Homologous recombination occurred in 6 of 114 clones, as indicated by the presence of both the wild-type (18.5 kb) and targeted (14.9 kb) alleles in KpnI-digested DNA. (b) SDS-PAGE analysis (Fairbanks’ method) of RBC membranes (5 μg protein per lane). Protein 4.2 is undetectable and band 3 decreased in 4.2^–/– RBCs. (c) Immunoblot analysis of RBC membrane ghosts (left; 5 μg protein per lane) and whole-cell lysates (right; 2.5 μL packed cells per lane). The blots were probed with a polyclonal antibody raised against purified human erythrocyte 4.2 (19). (d) Northern blot analysis of reticulocyte RNA from newborn mice (5 μg per lane). The filters were probed with a 2.4-kb mouse protein 4.2 cDNA (1). (e) Immunoblot of whole-platelet lysates probed with 4.2 antisera, as in c. Lane 1, 1.0 μg normal RBC membranes; lane 2, 3.0 μg normal platelet lysate; lane 3, 6.0 μg 4.2^–/– platelet lysate. (f) RNase protection assay of kidney mRNA. Perfused kidney mRNA (10 μg per lane) was hybridized with a 295-bp ³²P-labeled riboprobe corresponding to nucleotides 726–1019 (1) within the targeted region of protein 4.2 cDNA. In the left, middle, and right panels, RNase dilutions of 1:200, 1:100, and 1:50 were used, respectively. C is the RNA-free control lane. Sp, spectrin. Ank, ankyrin. bd 3, band 3. 4.1, protein 4.1. 4.2, protein 4.2. RBC, red blood cell ghost membranes. PLT, whole-platelet lysates.