Isolated splenocytes were starved in serum-free Iscove’s modified Dulbeco’s medium (IMDM) with 50 μM β-mercaptoethanol (Gibco, Life Technologies) for 1 hour. Subsequently, B lymphocytes were activated for 5 minutes at room temperature with H2O2 (4 mM) and anti-mouse IgM (10 μg/mL, 1022-01, Southern Biotech). Generation of whole-cell lysates and immunoblotting analysis were performed as previously.25 (link),27 (link) The antibodies used for western blotting were anti-actin (A5441, Sigma-Aldrich), anti-BTK (270-284, Sigma-Aldrich), anti-BTK pY551 (24a/BTK, BD Biosciences), anti-BTK pY223 (EP420Y, Abcam), anti-PLCG2 (Rabbit polyclonal, Biotech), and anti-PLCG2 pY753 (polyclonal, Abcam). The secondary antibodies, goat anti-mouse 800CW, goat anti-rabbit 800CW, goat anti-mouse 680LT, and goat anti-rabbit 680, were purchased from LI-COR Biosciences GmbH, Lincoln, NE, USA. Odyssey Imager from LI-COR Biosciences GmbH was used for membrane scanning, and the signals of total and phosphorylated proteins were quantified by NIH ImageJ 1.52a.
All experiments were approved by the local animal experimentation ethics committee, ID 1679. Mice were generated and maintained on C57BL/6 background. Analyzed Y223F mice and controls were sex- and age-matched. Experiments were performed on 7- and 22-week-old mice. Auto-antibody analysis was performed on 14- to 16-month-old animals.
All experiments were approved by the local animal experimentation ethics committee, ID 1679. Mice were generated and maintained on C57BL/6 background. Analyzed Y223F mice and controls were sex- and age-matched. Experiments were performed on 7- and 22-week-old mice. Auto-antibody analysis was performed on 14- to 16-month-old animals.
