Figure 4. Combination of anti-ErbB3 mAbs is more effective in inducing receptor internalization and degradation.

A. MST-L cells were treated with A3, A4 or their combination for 30′ and 1 h at 37°C in the presence of LysoTracker-Red as reported in materials and methods. Quantitative immunoflorescence analysis of the percentage of colocalization of signals and 3D reconstruction was performed as described in materials and methods. Results are expressed as mean values ± SE (standard errors): the percentage of colocalization was calculated analyzing a minimum of 50 cells for each treatment randomly taken from three independent experiments. Student's t-test was performed and significance level has been defined as described in materials and methods. A colocalization of ErbB3-bound A3, A4 and their combination with the LysoTracker marker in intracellular, perinuclear dots corresponding to lysosomes is evident. mAbs combination strongly accelerate receptor targeting to the lysosomal compartment compared to single treatments. **p < 0.001 vs. the corresponding A3 or A4-treated cells; the slight increase of colocalization of combination compared to single treatments at 1 h is not significant. Bar = 10 μm. B. Western blot analysis using anti-ErbB3 polyclonal antibodies in MST-L cells treated with A3, A4 or their combination at 37°C for the different time points (0.5, 1, 48 h). A drastic decrease of the band corresponding to ErbB3 is evident after mAbs combination treatments compared to single mAbs. The equal loading was assessed with anti-actin antibody and densitometric analysis was performed as described in materials and methods.