Multiimage 3 fc light cabinet

Protein lysates were separated by SDS-PAGE, blotted onto methanol-activated PVDF membranes (GE Healthcare, Buckinghamshire, U.K.) and incubated with primary antibodies against EGFR (D38B1, Cell Signaling, 1:1000), GAPDH (14C10, Cell Signaling, 1:1000), HLA-G (MEM-G/9, Exbio, 1:500), Notch2 (D76A6, Cell Signaling, 1:1000) and TCF-4 (C9B9, Cell Signaling, 1:1000) at 4 °C overnight. Finally, the blots were incubated with HRP-linked anti-mouse (1:25,000; GE-Healthcare) or anti-rabbit (1:5.000; Cell Signaling Technology, Danvers, MA) IgG secondary antibodies for 1 h at room temperature and developed with ECL Prime Western blotting detection reagent (GE Healthcare). The proteins were visualised with MultiImage III FC Light Cabinet and Alpha View 3.1.1.0 software (Alpha Innotech, San Leandro, CA).

Samples were lysed in Laemmli buffer and boiled for 5 min. Proteins were separated by using SDS-PAGE and blotted onto methanol-activated polyvinylidene difluoride membranes (GE Healthcare). After blocking in 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20, membranes were probed with the primary antibodies outlined in Table S1 overnight at 4°C, followed by incubation with horseradish-peroxidase-linked anti-mouse (1:25,000; GE Healthcare) or anti-rabbit (1:5000; Cell Signaling Technology) IgG secondary antibodies for 1 h at room temperature. The blots were developed with ECL Prime western blotting detection reagent (GE Healthcare), and proteins were visualized using MultiImage III FC Light Cabinet (Alpha Innotech) and Alpha View 3.1.1.0 software. For co-immunoprecipitation, approximately 5×10⁶ cells per condition were taken up in lysis buffer (catalogue number 9803, Cell Signaling Technology) supplemented with protease inhibitors (Sigma-Aldrich). The soluble fraction was incubated with a primary ErbB2 or ErbB3 antibody overnight at 4°C, and protein A agarose beads (catalogue number 9863, Cell Signaling Technology) were added for an additional 2 h. After five washing steps, samples were resuspended in Laemmli buffer and processed as described above.