Figure 3

LOXL2 expression in MCF10A cells increases phosphorylation of ErbB2. Cells were plated on a thin layer of Matrigel and serum-starved for 3 hours before being subjected to serum-blasting and subsequent lysis of cells. (A) Western blotting revealed that phospho-ErbB2 was elevated in 10A L2 cells when compared with 10A cont cells, whereas total ErbB2 levels were equivalent in the two lines, suggesting increased phosphorylation of ErbB2 in the 10A L2 cells. Densitometry analysis was calculated for pErbB2 levels relative to total ErbB2 and revealed a significant increase in 10A L2 cells (P = 0.0241). The levels of phospho-Akt and phospho-Erk1/2 were also elevated in 10A L2 cells. (B) The 10A cont cells were subjected to 16-hour treatment with 50 nM recombinant human LOXL2 (rhLOXL2; R&D Systems) followed by serum-starvation and serum-blasting (10A cont + rhLOXL2). Western-blotting analysis showed that in 10A cont treated with rhLOXL2, phospho-ErbB2 level was increased to a greater extent than 10A L2 cells when compared with sham-treated 10A cont cells. Densitometry analysis was calculated for pErbB2 levels relative to total ErbB2 and revealed significant increase in 10A cont + rhLOXL2 cells (P = 0.0438). This suggested that extracellular recombinant LOXL2 was capable of activating the ErbB2 receptor in 10A cells.