Nbp1 80695

Cells were first stained with Live/Dead Aqua (Thermo Fisher Scientific) in PBS for 10 min at 4 °C. Then, cells were stained in PBS containing 0.5% human AB serum and 2 mM EDTA with anti-CD1a APC and anti-CD16 FITC for 30 min on ice. After washing, cells were fixed with paraformaldehyde 4% in PBS for 20 min at room temperature and permeabilized with Permeabilization Buffer (Fixation/Permeablization Kit, BD Biosciences) containing Fc block (Human TruStain FcX, BioLegend) and mouse serum (BioLegend) for 30 min on ice. Cells were then incubated with the primary antibody in permeabilization buffer, rabbit anti-ETV6/Tel (Novus Biologicals, NBP1-80695, dilution 1:1,000) or rabbit anti-ETV3 (Atlas Antibodies, HPA004794, dilution 1:1,000), at 4 µg ml⁻¹ for 1 h at room temperature. Finally, cells were incubated with the secondary antibody anti-rabbit immunglobulin G (H + L) Alexa Fluor-594 (Molecular Probes, catalog no. A-11037, dilution 1:500) for 1 h at room temperature and then resuspended in staining buffer containing DAPI (Thermo Fisher Scientific, 50 ng ml⁻¹). Samples were acquired in an Amnis ImageStream instrument (Luminex). Data were analyzed using the IDEAS software to obtain the similarity score between DAPI and Alexa Fluor-594 channels. Finally, data were exported to FlowJo for quantification and visualization.

Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Fisher Thermo Scientific) supplemented with complete Mini EDTA-free protease inhibitor cocktail (Roche), at 1 × 10⁶ cells in 100 μl of lysis buffer. Postnuclear lysates were resolved by sodium dodecylsulfate–polyacrylamide gel electrophoresis using 4–15% BisTris NuPAGE gels (Invitrogen) and proteins were transferred to membranes (Immunoblot PVDF membranes, BioRad). Membranes were stained with primary antibodies against ETV6/Tel (Novus Biologicals, catalog no. NBP1-80695, 0.4 μg ml⁻¹), ETV3 (Atlas Antibodies, catalog no. HPA004794, 0.4 μg ml⁻¹), GP96 (Novus Biologicals, clone 9G10, 0.4 μg ml⁻¹) or actin (Millipore, clone C4, 0.4 μg ml⁻¹), followed by horeseradish peroxidase-conjugated secondary antibodies (Jackson Immunoresearch, dilution 1:10,000). Some membranes were incubated with Re-blot Plus buffer (Millipore). Densitometry quantification was performed using Fiji (v.2.9).