Fig. 6.
(a) Northern blot analysis of WNK4 in mouse. A segment of mouse WNK4 spanning exons 16-18 (GenBank accession no. AY_187027) was amplified from mouse kidney cDNA and verified by DNA sequencing. The segment was radiolabeled by random priming in the presence of 32P-labeled dCTP and hybridized to a mouse multiple tissue Northern blot containing poly (A)+ RNA (Clontech). After hybridization, the blot was washed and exposed to x-ray film. A 5.5-kb WNK4 transcript is most strongly expressed in kidney; transcripts are also detected in testis and heart. (b) Specificity of anti-WNK4 antibody. Full-length mouse WNK4 cDNA clone in pcDNA3.1– (1) was used for in vitro transcription/translation using the TnT-T7 Quick Coupled Transcription/ Translation System (Promega). In vitro-translated WNK4 was fractionated on 4–15% polyacrylamide gels by SDS/PAGE and transferred to nitrocellulose membranes. Blots were blocked, incubated with anti-WNK4 at 1:500, washed, and incubated with anti-rabbit IgG secondary antibody. Filters were washed and chemiluminesence was performed by using ECL+ (Amersham Pharmacia). The results (Left) demonstrate a band of ~135 kDa, the expected size of mouse WNK4 (2). Preincubation of the antibody with the immunizing peptide eliminated this band (Right).1. Wilson, F., Kahle, K., Sabath, E., Lalioti, M., Rapson, A., Hoover, R., Hebert, S., Gamba, G. & Lifton, R. (2003) Proc. Natl. Acad. Sci. USA 100, 680-684.
2.
Wilson, F., Disse-Nicodeme, S., Choate, K., Ishikawa, K., Nelson-Williams, C., Desitter, I., Gunel, M., Milford, D., Lipkin, G., Achard, J., et al. (2001) Science 293, 1107-1112.