Figure 2

Detection of the fusion gene product of the marker and β-actin. (a) Detection of β-actin::3xFLAG fusion gene product in HeLa cells by IP.The donor vector (0.5 μg) and ACTB-Cas9 or scr-Cas9 (0.5 μg) were transfected into HeLa cells in 3.5 cm dishes. Cells were harvested 72 hours after transfection. Lysates and proteins immunoprecipitated with anti-FLAG antibody were analyzed by WB. α-tubulin or IgG was used as a loading control for the input or IP samples respectively. (b) Detection of β-actin::3xFLAG fusion gene product in HEK-293T cells by IP. Samples were prepared in the same way as (a). (c) WB of RACK1::3xFLAG fusion protein in HeLa cells. Cells were harvested for WB 72 hours after transfection of RACK1-Cas9 and donor vectors. (d,e) Detection of β-actin::mClover fusion gene by FC and fluorescent microscopy. Error bars indicate standard error of the mean (SEM) of three independent experiments. Scale bar: 50 μm.