Supplemental Materials and Methods Immunofluorescence Cells (5x103 cells per chamber) were seeded into 8-chamber culture slides (BD Falcon, Franklin Lakes, NJ). The next day, cells were rinsed with ice-cold PBS and fixed with 4% paraformaldehyde for 10 min at room temperature followed by permeabilization with 0.1% Sodium Citrate plus 0.1% Triton X-100. The cells were subjected to immunofluorescence staining with ERBB2 (1:500) antibody for 2 h at room temperature. The cells were then washed with cold PBS three times for 3 min each, and incubated with Alexa 514-labeled anti-rabbit secondary antibody (1:800) (Invitrogen) at room temperature for 1 h. For dual immunofluorescence staining, cells were incubated with ERBB2 (1:500) and Flag (1:1000) antibodies for 2 h at room temperature. The cells were washed with cold PBS, and incubated with Alexa 568-labeled anti-rabbit (1:800) and Alexa 488-labeled anti-mouse (1:800) secondary antibodies (Invitrogen) at room temperature for 1 h. The cells were examined by fluorescence microscopy (Olympus America Inc, Center Valley, PA). Fluorescence intensities from images of six randomly selected microscopic fields of cells were semi-quantitatively analyzed by densitometry (ImageJ software, NIH Image). Cross-linking trastuzumab and cell surface ERBB2 receptor Cells (5x103 cells per chamber) were plated into 8-chamber culture slides (BD Falcon). To evaluate the interaction of trastuzumab with the ERBB2 receptor, cells were first treated with vehicle or trastuzumab (20 μg/ml) for 1 h on ice. To crosslink trastuzumab and ERBB2, cells were washed with PBS and incubated with BS3 (bis[sulfosuccinimidyl]) (5 mg/ml) (Thermo Scientific, Rockford, IL) in PBS for 30 min. Non-reacted BS3 was quenched with 20 mM Tris and cells were fixed with 4% paraformaldehyde for 45 min at room temperature. Without permeabilization, fixed cells were subjected to immunofluorescence staining with Alexa 488- labeled anti-mouse secondary antibody (1:800) and examined by fluorescence microscopy. Supplemental Figure 1. t-DARPP enhances ERBB2 expression on the cell surface A) OE19 cells stably expressing t-DARPP or pcDNA3 control vector were subjected to immunofluorescence staining with ERBB2 specific antibody as described in Supplemental Materials and Methods. B) ERBB2 fluorescence in control and t-DARPP- expressing cells was semi-quantitatively analyzed by densitometry. The results indicate that ERBB2 staining was mostly on the cell membrane, and ERBB2 expression was approximately 2 fold higher in t-DARPP-expressing cells than control cells (p<0.01). Supplemental Figure 2. t-DARPP decreases binding of trastuzumab to ERBB2 receptor A) OE19 cells stably expressing Flag-t-DARPP (OE19/t-DARPP) or pcDNA3 control vector were treated with trastuzumab and BS3 crosslinking reagent as described in Supplemental Materials and Methods. To evaluate the binding of trastuzumab to ERBB2 receptor, cells were subjected to immunofluorescence staining with anti-mouse secondary antibody that specifically reacts with trastuzumab. B) Trastuzumab/ERBB2 fluorescence in control and control and t-DARPP- expressing cells was semi-quantitatively analyzed by densitometry. The data indicate that trastuzumab-bound ERBB2 protein level was approximately 5 fold lower in t-DARPP expressing cells than control cells (p<0.01). C) In parallel, OE19/t-DARPP cells were subjected to dual immunofluorescence staining with Flag and ERBB2 antibodies to confirm the expression and localization of both t-DARPP and ERBB2 proteins. The results show strong cytosolic expression of t-DARPP that co-localized with the membranous ERBB2. The lower left cell colony that showed high levels of t-DARPP had higher levels of ERBB2 as compared to the upper right cell colony which had lower levels of t-DARPP and ERBB2. .