PMC

Cellular and surface expression of ErbB1-4 in L929 and cancer cells (a) Proteins were extracted from untransfected L929 cells, L929 cells transfected with erbB1-4 cDNA, or BT-474, SKBR3, and SKOV3 cancer cells and were probed with antibodies raised against ErbB1, ErbB2, ErbB3, ErbB4, and G6PDH (control).(b) Living cells were exposed to Sulfo-NHS-biotin to label surface proteins. Biotinylated proteins were subjected to Western blotting for ErbB2. Arrowheads marks the position of 175 kDa molecular marker and × represents the non-specific immunoreactivity that commonly appears in many types of cells.

Effects of neuregulin-1 on cell proliferation and binding to [³⁵S]-labeled neuregulin-1 (a) SKOV3 cells that were transfected with sulf-1 cDNA were serum-starved and then challenged with neuregulin-1 (10 nM or 40 nM).% ratios of changes in [³H] thymidine incorporation were calculated compared to that of untreated and mock-transfected SKOV3 cells.(b) Binding of [³⁵S]-labeled neuregulin-1 to SKOV3 cells was measured in the presence or absence of 40 nM cold neuregulin-1. (c) The effects of 40 nM neuregulin-1 on [³H] thymidine incorporation in BT-474 cells were assessed. (d) [³H] thymidine incorporation in parental L929 cells or ErbB2-L929 cells was determined. *p < 0.05, **p < 0.01, ***p < 0.001, compared to control cells.

Neuregulin-dependent and time-dependent decreases in ErbB2 phosphorylation (a) SKOV3 cells that had been transfected with sulf-1 cDNA and subjected to serum starvation were challenged with 40 nM neuregulin-1.Time-dependent alterations in ErbB2 phosphorylation at Tyr877, Tyr1139, or Try1196 as well as in authentic ErbB2 levels were examined by Western blotting. (b) Gel loading was controlled with β-actin levels. Changes in ErbB2 and ErbB3 phosphorylation were also assessed in BT-474. (c) Changes in ErbB2 phosphorylation were similarly assessed in ErbB2-L929 cells. *p < 0.05, **p < 0.01, ***p < 0.001, compared to the values of 0 time point (n = 3).

Total cell lysates (Total) and ErbB2 immunoprecipites (IP) were subjected to Western blotting with the anti-neuregulin-1 antibody (a) or anti-ErbB2 antibody (b). Prior to the electrophoresis, half of the immune complexes were treated with 1 M hydroxylamine to cleave the crosslinkage (lane 4). Arrowheads indicate the positions of molecular weight markers (kDa) Note; the cleavage of the crosslinker conversely elevated ErbB2 immunoreactivity (lanes 4 in b).

The tracer of neuregulin-1 was synthesized using the rabbit reticulocyte lysate system in the presence of [³⁵S]-methionine. [³⁵S]-labeled neuregulin-1 (1.7 kBq, 1.9 nM) was incubated in the presence of various concentrations of cold neuregulin-1 (0–40 nM). The ratios relative to the maximum binding with no competitor (100%) were determined in triplicate (n = 3). Data represent mean ± SE. Note; the total binding of the tracer was decreased to 54.3 ± 3.1% in parental L929 cells that were not exposed to cold neuregulin-1. The putative background binding found in parental L929 cells was not compensated as parental L929 cells express a low level of ErbB2.