Western blots were stained with antibodies specific for γ-secretase–cleaved NOTCH1 (Val1744; #4147 or #2421; Cell Signaling Technology), the intracellular transcriptional activation domain of NOTCH1 (Hasserjian et al., 1996 (link)), or the C terminus of NOTCH1 (#SC-6014 [C-20]; Santa Cruz Biotechnology). Control stains were performed with antibodies specific for actin (#ACTN05; Thermo Fisher Scientific), vinculin (#2907; Abcam), or GAPDH (#137179; Santa Cruz Biotechnology). Blots were developed with anti-rabbit HRP (#NA9340V; Amersham) or anti-mouse-HRP (#NA9310V; Amersham).
Expression of FR1 and FR2 was determined using isoform-specific antibodies (#MAB5646, R&D Systems; #103988, Abcam). Immunofluorescence staining was performed on permeabilized cells using a murine monoclonal antibody against NOTCH1 (3294, Abcam), and species-specific secondary antibodies linked to Alexa Fluor 488. Slides were mounted with Prolong Gold anti-fade reagents and counterstained with DAPI (Invitrogen). Images were acquired using a Zeiss LSM510 confocal microscope at 100× power. Cell surface NOTCH1 was evaluated by staining nonpermeabilized cells with monoclonal anti-human NOTCH1 antibody (#FAB5317P; R&D Systems) as previously described (Roti et al., 2013 (link)).
Expression of FR1 and FR2 was determined using isoform-specific antibodies (#MAB5646, R&D Systems; #103988, Abcam). Immunofluorescence staining was performed on permeabilized cells using a murine monoclonal antibody against NOTCH1 (3294, Abcam), and species-specific secondary antibodies linked to Alexa Fluor 488. Slides were mounted with Prolong Gold anti-fade reagents and counterstained with DAPI (Invitrogen). Images were acquired using a Zeiss LSM510 confocal microscope at 100× power. Cell surface NOTCH1 was evaluated by staining nonpermeabilized cells with monoclonal anti-human NOTCH1 antibody (#FAB5317P; R&D Systems) as previously described (Roti et al., 2013 (link)).
