In vitro biological activity of immune sera or purified immunoglobulins from rV- neu T vaccinated mice. Panel A: ADCC elicited by sera from 108 pfu rV-neu T vaccinated mice. SALTO tumor cells were exposed for 2 hours to sera pooled from rV-neu T or V-wt vaccinated mice and with mononuclear effector cells derived from normal BALB/c spleens at a ratio of 50:1. Results represent average percent cytotoxicity of two independent experiments. *p ≤ 0.01. Panel B: Effects of anti-Neu Igs on SALTO cell proliferation. SALTO tumor cells following serum depletion were incubated in DMEM medium containing 0.2% BSA with or without rV-neu T or V-wt purified Igs. Relative cell numbers of triplicate experiments were determined after incubation of 72 h at 37˚C using a sulforhodamine B based proliferation assay and expressed as percent increase or decrease in comparison to vehicle control (DMEM 0.2% BSA). *p ≤ 0.01. Panel C: p185 Neu receptor down regulation by anti-Neu Igs. SALTO cells were stained with stained with rV-neu T purified Igs and then with goat anti-mouse IgG Alexa fluor-488-conjugated antibody. Original magnification x 500. Expression and phosphorylation of ERK1 and ERK2 after rV-neu T Igs chronically treatment of SALTO tumor cells. Serum depleted SALTO cells were treated with rV-neu T- and V-wt Igs and the ratio between ERK1 and ERK2 and pERK1/pERK2 expression was analyzed by western blotting. Densitometric ratios between the phosphorylated and total levels of each protein are reported. Panel D: Effects of anti-Neu Igs on SALTO cells apoptosis. SALTO cells were plated at 2.5×104 cells/well, incubated in DMEM medium containing 0.2% BSA with or without rV-neu T or V-wt purified Igs (10 μg/ml) and stained with a specific anti-cleaved caspase 3 antibody. Staurosporine (1 μM) treatment was used as positive control. Nuclei were counterstained with Hoechst 33342. Original magnification x400.