Fig 2: IRCR201 suppresses c-Met signaling pathway via the degradation of c-Met. (a,b) A549 cells were pre-treated with dimethyl sulfoxide (DMSO) or MG132 (5 µM)/lactacystin (5 µM) mixture for 2 h and incubated with 100 nM anti-c-Met antibodies (IRCR201 or 5D5) or phosphate-buffered saline (PBS). After 4 h of incubation, the c-Met degradation pattern was measured by Western blot analysis. The band intensities of c-Met were quantified by a Multi Gauge V3.0 program and normalized by the intensity of corresponding ß-actin band; (c,d) A549 cells were pre-treated with DMSO or 100 nM concanamycin A for 2 h and subsequently treated with 100 nM anti-c-Met antibodies (IRCR201 or 5D5) or PBS for 4 h. The c-Met degradation pattern was evaluated by immunoblot analysis and analyzed by Multi Gauge V3.0 program; (e) Western blot analysis of c-Met, phospho-c-Met, Akt, phospho-Akt, Erk1/2, and phospho-Erk1/2 after 24-h treatment with IRCR201 (100 nM) or PBS in U87MG and MKN45; (f) Western blot analysis of c-Met, phospho-c-Met, Akt, phospho-Akt, Erk1/2, and phospho-Erk1/2 after 24-h treatment with IRCR201 (100 nM) or PBS in A549 in the presence or absence of HGF (50 ng/mL).