Extracellular matrix protein 1 activates extracellular signal-regulated kinase signaling by upregulating epidermal growth factor receptor and HER3. (A) At 24 hours after cell seeding, each cell line was treated with recombinant human extracellular matrix protein 1 (rhECM1; 200 ng/ml) or anti-ECM1 antibodies (5 μg/ml) and further incubated for 48 hours. Cells lysates were then analyzed by Western blotting. Cont, Control; ERK, Extracellular signal-regulated kinase; shC, Control short-hairpin RNA; shE, Extracellular matrix protein 1 short-hairpin RNA; TR, Trastuzumab-resistant; Vec, Vector; WT, Wild type. (B) Epidermal growth factor receptor (EGFR) and HER3 mRNA levels were determined by real-time PCR using primers specific for EGFR and HER3 (**P < 0.005, ***P < 0.0005). (C) Each cell line was treated with 100 μg/ml cycloheximide (CHX). Cell lysates were prepared at the indicated time points and analyzed on Western blots. Band intensities on the blots were quantified using 1DScan software (Scanalytics, Milwaukee, WI) and plotted versus time as the ratios of EGFR/actin and HER3/actin intensities (*P < 0.05).