Fig. 1.

Generation of gene-trapped insertional alleles for the mouse Nedd4 gene. (A) A gene-trapping vector was used to disrupt this gene into two independent cell lines: in the XA209 allele, the gene trap vector is inserted in the middle of the HECT domain (after exon 25), whereas in the XB398 allele, the gene trap vector is inserted after the first WW domain (after exon 12). (B) PCR genotyping of mice potentially harboring one of the alleles. (C) RT-PCR analysis of mice carrying the XA209 allele. Transcript containing exon 25 and the gene trap vector (exon 25-βGeo) was not detected in wild-type mice, whereas the endogenous transcript (exons 25/27) was not detected in mice homozygous for the XA209 allele. GAPDH was used as an internal control. (D) Immunoblot confirming the absence of Nedd4 protein in cells derived from XA209 homozygous mutants. Total cell lysate (50 µg protein) obtained from passage number–matched cultures was loaded in each lane. Primary antibody against the WW2 domain of Nedd4 (Upstate) and goat–anti-rabbit secondary antibody (Super Signal Femto, Pierce) were used.