Figure 5

Extracellular matrix protein 1 stabilizes epidermal growth factor receptor and HER3 proteins through galectin-3/mucin 1. (A) Lysates from each cell line were analyzed by Western blotting. shC, Control short-hairpin RNA; shE, Extracellular matrix protein 1 short-hairpin RNA; Vec, Vector; WT, Wild type. (B) At 24 hours after seeding, BT-474 trastuzumab-resistant (TR) cells were transfected with each small interfering RNA (siRNA: siC, Control; sigal-3, galectin-3; siMUC1, mucin 1), incubated further for 48 hours and analyzed on Western blots. (C) At 24 hours after seeding, cells were treated with recombinant human extracellular matrix protein 1 (rhECM1; 200 ng/ml) and incubated further for 48 hours. Cell lysates were then incubated with mucin 1 (MUC1), epidermal growth factor receptor (EGFR) and HER3 antibodies overnight. Immunoprecipitates (IP) were analyzed on Western blots. IgG, Immunoglobulin G. (D) Total cell lysates were incubated with MUC1, EGFR and HER3 antibodies overnight, and immunoprecipitates were then analyzed on Western blots.