Anti iβ3

To investigate association of membrane proteins, cells were lysed using a 1% Nonidet-P40 buffer containing a cocktail of protease and phosphatase inhibitors (Pierce). 400μg total protein lysate per tube was incubated overnight at 4°C under gentle end-over-end mixing with anti-TGF-βRII (1 μg, Santa Cruz). Subsequently, the immune complex was captured with protein A/G agarose resin, thoroughly washed with lysis buffer and eluted with non-reducing sample buffer. Proteins were then separated on a 10% SDS-PAGE gel (BioRad) and transferred to a PVDF membrane. Following blocking with 5% milk in TBS with 0.1% Tween-20, membranes were incubated with anti-Iβ3 (Santa Cruz, 1:1000) or anti-TGF-β RII (Santa Cruz, 1:1000) at 4°C overnight. After washing, membranes were blotted with anti-rabbit or anti-mouse IgG (1:5000 Santa Cruz), and bands were detected by enhanced chemiluminescence using an In Vivo MS FX Pro (Bruker). The Iβ3 and TGF-βRII co-precipitation was quantified by normalizing the band intensity of Iβ3 pulled down with TGF-βRII to total protein (anti-TGF-βRII). Analysis was performed using Image J software.

To investigate the association of TGF-β RII and Iβ3, we performed Förster Resonance Energy Transfer (FRET) and confocal microscopy. Briefly, the donor antibody (anti Iβ3 (SantaCruz)) was labeled with Alexa Fluor^® 488 Carboxylic Acid, Succinimidyl Ester (Life Technologies) (Iβ3-488) and the acceptor antibody (anti TGF-β RII (SantaCruz)) was labeled with Alexa Fluor^® 546 Carboxylic Acid, Succinimidyl Ester (Life Technologies) (TGF-β RII-546) in a 2.25:1 molar ratio of antibody:dye overnight at 4°. Labelled antibody was purified with size exclusion chromatography using PD-10 Desalting Columns (GE Healthcare). MDA-MB-231, RWGT2, and PC3 cells were grown on discs of rigid and compliant 2D PURs. After 24 hours of culture, cells were fixed with 10% Formaldehyde in PBS and stained overnight at 4° with Iβ3-488, TGF-β RII-546, Iβ3-488 + TGF-β RII-546 or IgG control (1ug/1×10⁶ cells). FRET experiments were performed on a BioTek Synergy 2 plate reader using excitation filter 485/20 and emission filter 590/35. Data were subtracted from the fluorescence of IgG control. Representative images of MDA-MB-231 cells grown on glass coverslips were taken with a Zeiss LSM 510 inverted confocal microscope. Images were obtained for Iβ3-488 (ex: 488, em: BP 505-550), TGF-β RII-546 (ex: 543, em: BP 560-615), colocalization (merge) and FRET (ex: 488, em: LP 585).