Fig. 6

U266 cells were serum starved and treated with the indicated concentrations of amuvatinib or DMSO and stimulated with 50 ng/ml HGF for 15 min. Cell lysates were subjected to immunoblot analysis. After transfer, the membrane was cut around 80 kDa and top portion was used for this figure while lower (<80 kDa was used for GAPDH as well as quantitation of proteins in Fig. 7d and e). To assess MET (Y1349) phosphorylation and total MET, the upper portion of the membrane was first probed with a rabbit anti-phospho-MET (Y1349) antibody followed by the secondary, which was a green fluorescent anti-rabbit antibody. Next, we probed with a mouse anti-total-MET antibody followed by the secondary, which was a red fluorescent anti-mouse antibody. The last antibody, the bottom portion of the membrane was probed with, was a mouse GAPDH antibody followed by the secondary red fluorescent anti-mouse antibody. We used a fluorescence based imaging system, LiCor Odyssey. The Odyssey software was used to convert color image to grey scale image for publication