Rabbit anti fli1

Protein was extracted from enriched primary splenic NK cells using Pierce RIPA buffer (Thermo-Fisher) with Halt protease inhibitor cocktail (Thermo-Fisher) and protein concentration was quantified using the Pierce BCA Protein Assay kit (Thermo-Fisher). Samples were electrophoresed on NuPage Novex 4–12% Bis-Tris Protein Gels, transferred to PVDF membranes, and blocked for one hour at room temperature with 5% w/v nonfat milk in 1X TBS and 0.1% Tween-20. Immunoblots were performed using rabbit anti-Fli1 (Abcam ab133485), rabbit anti-β-actin (Cell Signaling CST4970), and rabbit anti-GAPDH (Millipore Sigma G9545). Proteins were detected using the SuperSignal West Pico PLUS ECL kit (Thermo-Fisher) and visualized using the Azure Biosystems c280 imager. Band density was quantified using ImageJ version 1.53.

Immunoblot analyses were performed using standard protocols.^{17 (link)} Primary antibodies were used at the following concentrations: rat anti-HA (Roche, 1ug/ml), rabbit anti-FLI1 (abcam, 1ug/ml), and mouse anti-GAPDH (Millipore, 0.1 ug/ml). Secondary antibodies were goat anti-rabbit, goat anti-rat, and goat anti-mouse IgG respectively conjugated with horseradish peroxidase (Bio Rad, 1: 10,000 dilution). Membranes were developed using Western Lightning Plus-ECL enhanced chemiluminescence substrate (PerkinElmer) and visualized using photographic film.

ChIP assays of MSCs, SKNMC, A673 and HEK293T cells were carried out using two to five x 10⁶ cells per sample and per epitope, following the procedures described previously.^{51 (link)} In brief, chromatin from formaldehyde-fixed cells were fragmented to 200–700 bp with a Branson 250 sonifier. Solubilized chromatin was immunoprecipitated overnight at 4C with 3 mg of target specific antibodies (rat anti-HA (Roche), rabbit anti-FLI1 (Abcam), rabbit anti-H3K27ac (Active Motif), and rabbit anti-H3K9me3 (Abcam)). Antibody-chromatin complexes were pulled down with protein G-Dynabeads (Life Technologies), washed, and then eluted. After crosslink reversal, RNase A, and proteinase K treatment, immunoprecipitated DNA was extracted with AMP Pure beads (Beckman Coulter). ChIP DNA was quantified with Qubit. Sequencing libraries were prepared with 1–5 ng of ChIP DNA samples and input samples using the Ovation Ultralow System V2 kit (Nugen). Libraries were sequenced with single-end (SE) 50–75 cycles on an Illumina Nextseq 500 Illumina genome analyzer.