FIGURE 1. A novel Grb2-deficient cell line.

A, Predicted computer-generated stem-loop model using the RNAfold program (Vienna RNA Secondary Structure Prediction program web interface: http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) and the shRNA sequence from G⁺¹ to the termination signal UUUU of the RNA polymerase III (position +113) as input. As reference, Sal I and Xba I restriction sites are shown. B, transient transfection of COS7^WT cells with 2 mg of pU6⁺²⁷-shGrb2 or pU6⁺²⁷-shControl. COS7^WT cells were transfected and, at the times indicated, protein samples were obtained in order to monitor endogenous Grb2 protein levels by Western Blot. C, COS7^WT cells were transfected with the indicated amounts of pU6⁺²⁷-shGrb2 or pU6⁺²⁷-shControl and samples analyzed by Western Blot. D, Generation of stable cell lines. COS7 cells were stably transfected with pU6⁺²⁷-shGrb2 or pU6⁺²⁷-shControl as indicated in Methods. Forty clones resistant to neomycin and expressing pU6⁺²⁷-shGrb2 were screened for Grb2 expression by Western blot. The process was repeated a total of three times. In the end, 3 subclones were stocked and used for further studies. Shown is the expression of Grb2 in comparison with that of original cells.