Confirmation of target gene expression using Robo1 as an example. (A). Loss of Robo1 expression in the RHGP clone whose robo1 gene was perturbed by RHGP in the presence of RSL1 (on) and reemergence of expression in the absence of RSL1 (off). Cell lysates of WT MT4 cells and RHGP clone were resolved in an SDS-PAGE gel. After transfer to a membrane, the blot was first probed by anti-Robo1 (A301-265A, Bethyl Laboratories) and then anti-HSP90 (heat shock protein) after stripping, each followed by 2nd HRP-conjugated anti-rabbit Ab. (B). Knockdown expression of Robo1 in MT4 cells treated with siRNA against robo1. Cell lysates from samples treated with different siRNA as indicated were processed into western blot as described above. Herein GAPDH protein probed by an anti-GAPDH was used as a loading control.