Fig. 1.
PCR and Western blot analyses of F6 generation mice. A: Top: PCR analysis of tail DNA from the same litter identifying two wild-type (WT) (+/+), one heterozygous (+/−), and one Lasp-1 knockout (KO) (−/−) mouse based on criteria described in materials and methods. 100-bp DNA ladder (Invitrogen) is included in the first lane. Bottom: Western blots of gastric mucosal extracts from the same mice showing differences in the Lasp-1 signal (8C6 antibody; 1:2,000 dilution) compared with the β-actin signal (1:5,000 dilution). B: quantitation of Western blot signals from gastric mucosal extracts from F6-F10 generation littermates obtained with antibodies directed against β-actin and several endocytosis-associated proteins. Antibody dilutions were as follows: dynamin, 1:1,000; Huntington Interacting Protein 1 Related (Hip1r), 1:1,000; β-actin, 1:5,000; clathrin heavy chain, 1:1,000; zyxin, 1:500. Values from Lasp-1-null mice expressed as a percentage of band densities from WT littermates. N = 5–9, P > 0.1. C: Western blot analyses of Lasp-1 and Lasp-2 expression in gastric mucosa and brain. Left: Lasp-1, which migrates on SDS PAGE gels with an apparent molecular ratio (Mr) of ∼37 kDa, was detected in both tissues. Lasp-2 (LIM-nebulette), which migrated with an apparent Mr of ∼30 kDa, was detected only in brain. Right: in littermate comparisons, no Lasp-1 expression was detected in gastric mucosal extracts from Lasp-1−/− mice. In other experiments, signals were quantified and found to be ∼50% lower in Lasp-1+/− compared with Lasp-1+/+ mice (not shown). Lasp-2 was not detected in any of these extracts.