Extracellular matrix protein 1 augments epidermal growth factor signaling. (A) At 24 hours after seeding, BT-474 trastuzumab-resistant (TR) and BT-474 extracellular matrix protein 1 (ECM1)-expressing cells were treated with anti-ECM1 antibodies (ab; 5 μg/ml). Ten minutes later, cell lysates were analyzed on Western blots. (B) After serum starvation for 24 hours, cells were treated with recombinant human extracellular matrix protein 1 (rhECM1; 200 ng/ml) and epidermal growth factor (EGF; 10 ng/ml). Cell lysates were prepared at the indicated time points and analyzed on Western blots. (C) Total cell lysates were incubated with epidermal growth factor receptor (EGFR) antibodies overnight, and immunoprecipitates (IP) were analyzed on Western blots. IgG, Immunoglobulin G; shC, Control short-hairpin RNA; shE, Extracellular matrix protein 1; Vec, Vector. (D) Cells were incubated with 0.5 mg/ml EZ-Link NHS-SS-Biotin for 30 minutes at 4°C. The biotinylated proteins were precipitated by streptavidin, and the precipitates were analyzed on Western blots (IB) using ECM1 antibody (left). Cell surface labeling of ECM1 was conducted by immunostaining without permeabilization (right). Extracellular signal-regulated kinase (ERK) was used as an endogenous negative control protein. DIC, Differential Interference Contrast; GSH, Glutathione.