Irf4 antibody

10 cm dishes were seeded with 4×10⁶ 293T and were transfected the next day using TransIT-293T (Mirus). 48 hours later cells were washed 2 times with ice cold PBS, and lysed while rocking at 4°C, in 1 mL Triton X Lysis Buffer (50 mM Tris HCl pH 7.4–7.5, 150 mM NaCl, 1 mM EDTA, 0.1% Triton; supplemented with 1 mM NaF, 1 mM activated Na3V04, and Roche EDTA free protease inhibitor cocktail tablet for 50 mL volume). Following lysis, membranes were pelleted and lysates were transferred to pre-chilled tubes. Protein concentration was determined using DC Protein Assay (BioRad). 1 mg of cell lysate was precleared with prepared protein G beads (Pierce), then 8 ug of IRF4 antibody (clone M17, Santa Cruz Biotech) or flag antibody (clone M2, Sigma) was added and lysates were incubated overnight at 4°C. Lysates were transferred to freshly prepared protein G beads for binding and were incubated at 4°C for 2 hours. Following wash, protein was eluted and samples were electrophoresed on 10% polyacrylamide gels, and transferred onto nitrocellulose membranes for western blot. The following detection antibodies were used: IRF4 (clone H140, Santa Cruz Biotech.) and Flag (clone M2, Sigma).

FACS-sorted cells were homogenized in lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, and 1 μg/ml leupeptin (Cell Signaling Technology) supplemented with 1% Halt protease inhibitor cocktail (Thermo Fisher Scientific). Protein concentrations were then quantified using a Bio-Rad protein assay kit (Bio-Rad Laboratories). A total of 15 µg of each protein lysate from mCherry-positive cells of the Tg(lck:IRF4) fish were mixed with 4× Laemmli sample buffer (Bio-Rad Laboratories) and β-mercaptoethanol (Sigma-Aldrich) in appropriate proportions before being incubated at 99 °C for 5 min. The samples were then separated on SDS-PAGE gels and transferred to PVDF membranes (Millipore) at 400 mA for 1.5 h, blocked with 3% skim milk (Nacalai Tesque) in Tris-buffered saline with 0.1% Tween and probed with IRF4 antibody (1:1000; Santa Cruz), cleaved PARP antibody (1: 1000; Cell Signaling Technology) and GAPDH-HRP antibody (1: 1000; Santa Cruz). Secondary detection was performed with HRP-linked antirabbit IgG (Cell Signaling Technology) and ECL™ Western blotting detection reagents (Fisher Scientific). Western blot images were captured using an ImageQuant LAS500 chemiluminescent image analyzer (GE Healthcare).