Figure 4.

Multiple MAPK effector molecules are inhibited in Ctx^R clones by Sym004 treatment. (A) Sym004 inhibited multiple downstream MAPK effector molecules detected through phospho-kinase array. After treatment with Sym004 (50 µg/ml), cells were collected and cell extracts were incubated with membranes containing antibodies to 26 individual proteins. The membranes were washed and incubated with a cocktail of biotinylated detection antibodies, streptavidin-HRP, and chemiluminescent detection reagents to measure the levels of phosphorylated protein. Quantitation of phosphorylated proteins was completed using scanned images from ImageJ software. Data points are represented as the mean of duplicate spots. Cell extracts were also fractionated on SDS-PAGE, followed by immunoblot analysis for the indicated proteins. α-Tubulin was examined as a loading control for each immunoblot analysis. (B) Sym004 can induce a G1-phase cell cycle arrest in Ctx^R clones. Cells were treated with 20 µg/ml Sym004 for 24 hours, and cell cycle phase distribution was analyzed as described in the Materials and Methods section. Data points are represented as means ± SEM (n =3). *P < .05.