Knockdown Effects on Class Switch Recombination

Class switch recombination was performed in CH12F3-2a cells as described previously^{50 (link)}. Briefly, RNF8, RNF168, L3MBTL2 or a combination of these, was knocked down using shRNAs. 40 hours later, cells were stimulated with ligands [1 ng/ml of recombinant human TGF-β1 (R&D Systems), 10 ng/ml of recombinant murine IL-4 (R&D Systems), and 250 ng/ml recombinant murine CD40 ligand (PerproTech)]. For analyzing class switch from IgM (IgM+/IgA−) to IgA (IgM−/IgA+), CH12F3-2 cells were collected after 60 hours, intracellularly stained with PE-conjugated anti-murine IgA antibody (clone 11–44-2, eBiosciences, Cat# 12-5994-82). Membrane IgM expression was assessed using FITC-conjugated anti-murine IgM antibody (eBiosciences; Cat# 11-5890-82). Cytofix/Cytoperm and Perm/Wash buffers (BD Biosciences, catalog # 554714) was utilized. Cells were then analyzed on a FACS Calibur (BD Biosciences) at the Mayo Clinic Flow Cytometry Core. Data was analyzed using FlowJo software (TreeStar).