FIG. 4.

Construction of heterozygous (PAP^+/−) DT40 cell lines. (A) A restriction map of the chicken PAP locus and the knockout constructs neo-PAP and hygro-PAP. The bacterial neomycin- or hygromycin-resistant genes were inserted into exon 2 (indicated by a solid box) of the PAP gene. The restriction sites are indicated by arrowheads and a single capitalized letter: E (EcoRI), K (KpnI), and X (XbaI). (B and C) Southern blot analyses of genomic DNA isolated from wt, kh12 (hygro-PAP targeted), and kn1 (neo-PAP targeted) cells. DNA (30 μg) was digested with EcoRI (B) or EcoRI and XbaI (C) and then separated on a 7% agarose gel. The positions of the DNA size markers are indicated in kilobases on the left. (D) Western blot analyses of whole-cell extracts isolated from wt, kh12, and kn1 cells. Affinity-purified PAP polyclonal antibodies were used (upper panel). The same filter was probed with mAb104 monoclonal antibodies (recognizing splicing factor SRp75 and other proteins) to confirm equal loading of extracts in each lane. The positions of PAP and SRp75 are indicated by arrows. The positions of protein size markers are indicated in kilodaltons on the left.