Targeted disruption of the Lsc gene eliminates Lsc expression. (A) Schematic diagrams of the native Lsc gene locus, Lsc targeting vector, and targeted Lsc gene locus. Shaded boxes indicate translated exons; dashed lines, boundaries of recombination between the native locus and targeting vector; solid triangles, orientation of the neomycin resistance (NeoR) and the thymidine kinase (TK) genes. (B) Southern blot of tail genomic DNA from the progeny of an Lsc^-/+ mating, with the 3′ probe that hybridizes to a 9.6-kilobase (kb) fragment released from the native locus and a 6.6-kb fragment released from the targeted locus. Size markers at right are kb. (C) RT-PCR products generated from splenocyte oligo dT-primed total RNA with primers for Lsc and GAPDH. Size markers at right are base pair (bp). (D-E). Western blots of splenocyte (D) and neutrophil (E) lysates with anti-Lsc antibody. Multiple bands in Lsc^+/+ neutrophil lysates may be Lsc splice variants or degradation products. Molecular weight markers at right, kDa. (F) Confocal images of Lsc KO neutrophils prepared and labeled as described in Figure 1. Bars represent 10 μm.