Fig. 1

ABT-700 specifically binds cellular c-Met and antagonizes c-Met signaling in both HGF-dependent and -independent settings. a FACS analysis of ABT-700 binding to MCF7 transfectants. Stable human c-Met or vector control transfectants of human MCF7 breast cancer cells were incubated with increasing amounts of ABT-700 and bound ABT-700 was detected by FACS with secondary anti-human IgG conjugated with Alexa 488. b ELISA quantification of phospho-c-Met in A549 cells. A549 cells grown in a 96-well plate were pre-incubated for one hour with antibodies in a dose-range as shown, followed by stimulation with 1 nM HGF for 10 min. Total cell lysates were made and phospho-c-Met was detected by ELISA. c ELISA quantification of phospho-c-Met in SNU5 cells. SNU5 cells grown in a 96-well plate were incubated with antibodies in a dose-range as shown for 6 h. Total cell lysates were made and subjected to ELISA for phospho-c-Met. The value of cells in media alone was used as 100 % of control. d ELISA quantification of total c-Met in SNU5 cells. SNU5 cells grown in a 96-well plate were incubated with antibodies in a dose-range as shown for 6 h. Total cell lysates were made and c-Met level was determined by ELISA. The value of cells in media alone was used as 100 % of control. e Western blot analysis of U87MG cell lysates. U87MG cells grown in a 12-well plate were treated with antibodies as shown at 10 μg/mL for 10 min, 1 h or 6 h. Total cell lysates were analyzed for c-Met and other phosphorylated targets as shown. Western blot analysis of Hs746T cell lysates. Hs746T cells grown in a 12-well pate were treated with antibodies as shown at 10 μg/mL for 6 h. Total cell lysates were analyzed for c-Met and other phosphorylated targets as shown. f Western blot analysis of SNU620 cell lysates. SNU620 cells grown in a 12-well pate were treated with antibodies as shown at 10 μg/mL for 24 h. Total cell lysates were analyzed for c-Met and other phosphorylated targets as shown. g Inhibition of proliferation of SNU620 cells. SNU620 cells were plated in a 96-well plate and treated with antibodies in a dose range as shown for 3 days. Quantification of live cells at the end of incubation was done with Cell-titer Glo reagents. The data shown in all panels are from one of at least two independent experiments showing similar results as described in Methods