Fig. 1: R2 evicts WTErbB-2 and ErbB-2c from the nucleus of BC cells.

A Left: Representative WB of ErbB-2 expression. Right: signal intensities of ErbB-2c and WTErbB-2 were analyzed by densitometry from three independent WBs performed as indicated. Fold change was calculated by normalizing the absolute levels of each ErbB-2 isoform to those of β tubulin, setting the value of vehicle-treated cells to 1. B Representative WB of cell lysates with the indicated phosphospecific or total antibodies. C, D ErbB-2 immunofluorescence (IF) in cells treated with R2 or vehicle (24 h). Bottom panels: quantitative analysis of ErbB-2 subcellular localization. Fluorescence intensity of nuclear, cytosolic, and membrane ErbB-2 was quantified and is plotted as percentage (mean ± SD, n = 50 per group) relative to total ErbB-2 in each cell. E Inhibition of NErbB-2 localization in cells from C and D (mean ± SEM). For c and d vs a: P < 0.001; for c and a vs b: P < 0.01, for d vs b: P < 0.05. F Nuclear and cytosolic lysates were analyzed by WB. Fold change was calculated for each compartment by normalizing ErbB-2 levels in treated vs control cells (value set to 1). G ErbB-2 and Stat3 were localized by IF and confocal microscopy in cells treated as in D. Merged images show colocalization in yellow. The insets show boxed areas in detail. Right: Quantitative analysis of colocalization with Manders’ coefficients (M1 and M2, mean ± SEM, n = 50 per group). H Subcellular distributions of AR and c-Jun evaluated as in F. I, J Cells were pretreated with R2 (100 µM) or vehicle for 24 h and then treated with HRGβ1 (60 min) (I), or MPA (90 min) (J). ErbB-2 localization and NErbB-2 levels are depicted as in C. ns: not significant, *** P < 0.001. For (A–J), n = 3. Original uncropped WB images are shown in Fig. S9.