144 | Chapter 5 Supplemental materials and methods Immunoblotting For whole-cell lysates, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl at pH 7.0, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, and 1% NP40), supplemented with EDTA-free complete protease inhibitor cocktail and phosphatase inhibitor cocktail (PhosSTOP™) (both from Roche, Basel, Switzerland). For cytoplasmic and nuclear fractionation, the Nuclear/Cytosol Extraction kit was used according to the manufacturer’s instructions (Biovision, Mountain View, CA). Equal amounts of lysates (10 µg) were separated on 4% to 20% gradient SDS page gels (Bio-Rad, Hercules, CA) and transferred to PVDF membranes (Fisher Thermo Scientific, Waltham, MA). Antibodies used for immunoblotting include RAG1 (D36B3), FOXO1 (C29H4), p-FOXO1-ser-256 (9461), FOXO3a (75D8), p-AKT-ser-473 (D9E) (Cell Signaling Technology, Danvers, MA), p-FOXO1- ser-329 (EP927Y), C-MYC (Y69), KU70 (N3H10) (Abcam, Cambridge, MA), IκBα (SC-371), p-IκBα-ser-32 (SC-8404), p-IKKα-thr-23 (SC-101706), NF-kB p65 (SC-372), CDK4 (SC-260), p-STAT5-tyr-694/ tyr-699 (SC-11761) (Santa Cruz Biotechnology Inc. Dallas, TX), and β-Actin (AC-15, Sigma Aldrich). Flow cytometry Intracellular, intranuclear and BrdU stainings were done as previously described1,2. Antibodies for mouse cells include B220-APC (RA3-6B2), CD43-PE (S7), kappa light chain-PE/ Cy7 and kappa light chain-FITC (187.1) (BD Pharmingen, San Diego, CA), IgM-PE/Cy7 (II/41), p-H2AX-ser-139- Alexa-Fluor-647 (2F3) (eBioscience, San Diego, CA). Antibodies for human cells include CD19-APC/Cy7 (BD, Pharmingen) CD43-FITC (clone 581, Biolegend, San Diego, CA), CD10-PE/Cy5 (BD Pharmingen) and IgM-APC (BD Pharmingen). Intracellular Ig kappa light chains stainings and intranuclear γH2AX stainings were performed as described earlier1,2. BrdU incorporation was measured by incubating cells for 2 hours with 20 µM BrdU (Sigma Aldrich), BrdU stainings were done as described previously1,2. All populations were gated on viable cells by 7-aminoactinomycin-D exclusion or based on forward and side scatter. Doublets were excluded based on forward and side scatter height versus width profiles. Flow-cytometry was performed on a four-laser, thirteen-detector LSR Fortessa (BD Biosciences). Flowcytometry data was analyzed using FlowJo (Tree Star Inc., Ashland. OR). Analysis of Jk2-RSS signal end DNA breaks in G1- and S- cell-cycle phase cells WT and RAG2-/- Abl transformed pre-B cells were treated with 2.5 mM IKKβi + AKTi or left untreated for 48h and subsequently stained with 10 µM Vybrant® Dyecycle™ (Life Technologies, Carlsbad, CA) for 90 minutes at 37°C. G1- phase and S-phase cells were sorted according to their DNA content using a BD Influx™ cell-sorter (Becton & Dickinson. Exactly 1x106 cells were sorted for each fraction, cells were washed and embedded in 0.5% low-melting
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