Anti maml3

Co-IP was performed in all three cells lines using standard protocols. In brief, two days post-transfection, FL MAML3 and β-catenin co-transfected cells were crosslinked with 1% formaldehyde and quenched with glycine. Cells were harvested on ice in lysis buffer: 5 mM PIPES pH, 8 85 mM KCL, 0.5% NP-40 and Roche protease inhibitors (5892791001) and then incubated with protein-A sepharose resin (Invitrogen 101041). Lysates were immunoprecipitated overnight at 4°C with either rabbit IgG (Cell Signaling 2729) or anti-MAML3 (Bethyl Laboratories A300–684) on protein-A sepharose blocked with 3% BSA. Resins were spun at 500g for 5 minutes, flow through discarded, and then washed 2X with 10 resin-volumes lysis buffer followed by 3X washes with 500 mM LiCl. Proteins were eluted from the column by boiling in Laemmli buffer for 30 minutes. Samples were visualized by immunoblot probed with anti-β-Catenin D10A8 (Cell Signaling Technologies, 8480).

Nuclear protein analysis was performed as previously described (44 (link)) using anti-Notch1^val1744 (Cell Signaling Technology, 4147S), anti-Notch2 (Cell Signaling Technology, D76A6), anti-Notch3 (Cell Signaling Technology, D11B8), anti-Notch4 (Cell Signaling Technology, L5C5), anti-MAML1 (Cell Signaling Technology, D3K7B), anti-MAML2 (Cell Signaling Technology, D41E6), anti-MAML3 (Bethyl Laboratories, A300–6841), and anti-RBPSUH (CSL, Cell Signaling Technology, D104A).