Regulation of ErbB signalling by chronic Morphine in SKBR3 cells.

<p>(<b>A</b>) Identification of κ-opioid receptors in SKBR3 human mammary adenocarcinoma cells by RT-PCR. Reactions contained 10 ng of cDNA from SKBR3 cells or plasmid pcDNA3.1 containing the cloned receptor. A 322 bp fragment of the κ-opioid receptor is present in the cells (arrow). Ladder: 100 bp. (<b>B</b>) Regulation of ErbB receptor abundance by chronic Morphine treatment. Cells were cultured for 3 d in the presence or absence of Morphine (10 µM) and Naloxone (100 µM), before membranes were prepared and ErbB receptors were determined by Western blot using antibodies specific for ErbB1 (175 kDa), ErbB2 (185 kDa) and ErbB3 (185 kDa). Equal protein loading was determined using an antibody against β-tubulin. (<b>C</b>) Effect of chronic Morphine treatment on ErbB1- and ErbB3-stimulated ERK1/2 signalling. SKBR3 cells were cultured for 3 d in the absence (control) or presence of Morphine (10 µM), before the impact of protein inhibitors AG1478 (ErbB1), AG825 (ErbB2), Wortmannin (Wort; PI3K) and EGCG (metalloproteinases) on basal, EGF (100 ng/ml)- and Heregulin (40 ng/ml)-stimulated ERK1/2 phosphorylation was determined by Western blot. Controls were kept in the absence of protein inhibitors (none). Total ERK1/2 was determined using an overall reactive antibody.