Second-site mutation in the Wiskott-Aldrich syndrome (WAS) protein gene causes somatic mosaicism in two WAS siblings
J. Clin. Invest. Taizo Wada, et al. 111:1389 doi:10.1172/JCI15485 [Go to this article.]

Figure 6
In vitro reconstitution experiments. (a) Retrovirus-mediated expression of WASP. Western blot analysis of WASP was performed using lysates of WAS T cell lines transduced with the indicated retroviral vectors. Arrows indicate the position of the WASP bands. (b) Analysis of the inserted proviral genome by semiquantitative PCR. WASP exons 3 and 4 were amplified using DNA obtained from WAS T cell lines transduced with the indicated retroviral vector. The ratio of the band intensity of the endogenous WASP gene and the inserted transgene is shown. (c) In vitro binding of WASP to SH3 domain-containing proteins. Lysates were incubated and precipitated with SH3-GST fusion proteins. Complexes were then resolved by SDS-PAGE, and WASP was detected by Western blot analysis. (d) Analysis of TCR/CD3 downregulation. T cells were subjected to CD3 cross-linking at 37°C for 60 min and then stained with PE-conjugated streptavidin to analyze CD3 expression. Data are expressed as the percentage of mean fluorescent intensity of unstimulated control cells and represent the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01. UT, untransduced; Neo, Neo control vector; IB, immunoblotting; End, endogenous; Tg, transgene.