NOTCH3 protein, human

In all, 40 × 10⁶ MDAMB-231 cells were seeded 24 h before treatment or not with 1 μg/mL of doxycycline to induce the intracellular domain of Notch3 (N3ICD). Cells were scraped, centrifuged at 800 × g for 5 min and washed with 20 mL of cold PBS before fixation in 20 mL of paraformaldehyde (PFA) for 10 min at 4 °C. Cells were washed three times with cold PBS, resuspended in 9 mL of L1 buffer (50 mM Tris pH 8.0, 2 mM EDTA, 0.1% NP40, 10% Glycerol) for 5 min on ice. Cells were centrifuged at 2000 × g at 4 °C for 5 min and the pellet resuspended in 2 mL of L2 buffer (50 mM Tris pH 8, 5 mM EDTA, 1% SDS). The chromatin fragmentation was done by sonication at 20%, for 6 min with 1 s between each pulse then the debris were removed by centrifugation at 9400 × g for 5 min. DNA concentration was assessed by collecting 150 μL of fragmented DNA and mixing it with 2 μL of proteinase (10 mg/mL) and 6 μL of NacL (5 mM) for 30 min at 37 °C. Then, 2 μL of proteinase K (20 mg/mL) was added and incubated at 65 °C for 2 h. DNA was purified with NucleoSpin Gel and PCR clean-up (MN 740609.5), and 10 μg of DNA was diluted in 9 volumes of dilution buffer (20 mM Tris pH 8.0, 2 mM EDTA, 0.1% SDS, 1% NP40, 500 mM Nacl). 100 μL served as input. A pre-clear was conducted by adding of 30 μL of chip-Grade Protein G Magnetic Beads (Cell signaling) for 3 h at 4 °C. The supernatant was collected and 2 μg of antibody HeyL or HA (sigma-aldrich) was added and incubated overnight at 4 °C. To collect the immunocomplexes, 30 μL of beads were supplemented to the mix DNA/antibody and incubated for 30 min at 4 °C then centrifuge at 3400 × g for 1 min. Immunocomplexes were washed three times for 5 min with washing buffer (20 mM Tris pH 8.0, 2 mM EDTA, 0.1% SDS, 1% NP40, 500 mM NaCl) and three times with 1x TE buffer. The Ag/Ab complexes were extracted in 100 μL of elution buffer (1x TE buffer, 2% SDS) and reversion of crosslinking was done with proteinase A, NaCl and proteinase K then DNA purified with Nucleospin Gel and PCR clean-up. A q-PCR was then performed on input and Chip using the following primers:
MYBL2.1prom-F: AGGAGAGGAAGCAGGGAGAG
MYBL2.1prom-R: CATAGCGAAGACCGAGGAAG
MYBL2.2prom-F: TTTTGTCTCCCGCCTAATTG
MYBL2.2prom-R: CCGGAATGTTAAGGAGCAAA
HEYLprom-F: GCTCTCATGCAGCTTCCTTT
HEYLprom-R: GGCAACCCATCAAACTGTTC
HEYLprom4F: CATTACTGCATCTTCCCCGC
HEYLprom4R: AGACGTTGGCTCTGAGTTGA
HEYLprom5F: ACATACCCCAACTCTGCTCC
HEYLprom5R: TTGGCTCGCAACAAATCCAA
HEYLprom6F: CAAGACCCCACTGTGATCCT
HEYLprom6R: GCTGGGGTTGTTGTGTCTTT
HEYLprom7F: TAGTCAGTGAGAGGGTGGGT
HEYLprom7R: ACATAGTGTCTGCCTCGCTT

Immunohistochemistry was performed on 4-μm-thick sections of formalin-fixed, paraffin-embedded and heat-treated (for antigen retrieval) tissues (DakoCytomation). Sections were stained with hematoxylin-eosin-safran and with anti-Notch3 (Cell signaling, D11B8) anti-CD31 (anaspec (53332)), anti-Ki67 (eurobio, ref M3064). Positive cells were counted on whole sections using HALO software. Investigators were blinded when analyzing immunohistochemistry staining with Halo software.