Anti myc antibody

Cells were fixed for thirty minutes in 4% paraformaldehyde + 1% glutaraldehyde and blocked for thirty minutes in a 5% normal donkey serum, 0.2% saponin, PBS solution. Cells were incubated overnight at 4°C with an anti-myc antibody (Developmental Studies Hybridoma Bank, 9e10), followed by secondary staining using Alexa Fluor secondary anti-mouse antibody (Thermo). Anti-Myc (9e10) was deposited to the DSHB by Bishop, J.M. (DSHB Hybridoma Product 9e10). Actin staining was performed using Alexa Fluor 647 Phalloidin (Thermo) according to manufacturer instructions. Micrographs were taken using an AMG EVOS microscope.

The plasmid used for the mass spectral analyses were pSecTag2c-mouse NOTCH1 EGF1-36-Myc-His₆, mouse NOTCH1 EGF1-18-Myc-His₆, mouse NOTCH1 EGF19-36-Myc-His₆ or mouse NOTCH1 EGF24-28-Myc-His₆ [26 (link)], and pSecTag2c-mouse NOTCH2 EGF1-36-Myc-His₆ [23 (link)]. HEK293T cells (7.0 × 10⁶) were seeded in a 100/20 mm cell culture dish in DMEM, 10% calf serum, and transiently transfected with 1 μg/well plasmids, using PEI. After 4–5 h, the medium was changed to 6 mL of OPTI-MEM I. The cells were cultured for another 3 days. For the purification of the Notch proteins, the media samples from the transfected cells were applied to Ni-NTA agarose (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) affinity chromatography using Poly-prep gravity-flow columns (Bio-Rad, Hercules, CA, USA). After washing with TBS (10 mM Tris buffer pH 8.0 containing 150 mM NaCl) containing 10 mM imidazole, the bound proteins were eluted with TBS containing 250 mM imidazole. The purified samples were analyzed by Western blotting using the anti-Myc antibody (Developmental Studies Hybridoma Bank (DSHB), University of Iowa, Iowa City, IA, USA, 1:2000 dilution) and GelCode blue stain (Thermo Fisher Scientific, Waltham, MA, USA).