Anti irf4

Five-7 x 10⁶ cells were prepared by lysis in a buffer made up of 20 mM Tris, 150 mM NaCl, 2 mM EDTA (ethylenediaminetetraacetic acid), 2 mM EGTA (ethylene glycol-tetra-acetic acid), 0.5% v/v Triton X-100 supplemented with protease inhibitor cocktail (Sigma), 1 mM DTT (dithiothreitol; Amersham Biosciences), 1 mM PMSF (phenyl-methyl-sulfonyl fluoride; Sigma), 1 mM okadaic acid (Sigma) and phosphatase inhibitor cocktail (Thermo Scientific). Proteins were subjected to SDS-PAGE, transferred to PVDF membranes and immunoblotted with the following primary Abs: anti-CK2α (provided by Dr. M. Ruzzene, University of Padova, Italy), anti-CK2β, anti-RELA, anti-FOXO1 (Abcam), anti-pRELA S529 (recognizes S527 in mouse), anti-IRF4, anti-BLIMP-1, anti-BCL6 (Santa Cruz), anti-GAPDH (Ambion), anti-pAKT S129 (provided by Dr. M. Ruzzene, University of Padova, Italy), anti-AID (Invitrogen), anti-NOTCH2 (D76A6), anti-pAKT S473, anti-AKT, anti-pERK1/2 T202/Y204, anti-pBTK Y223, anti-ERK1/2, anti-pPTEN S380/T382/T383, PTEN, anti-pFOXO1 S256, anti-pSTAT6 Y705, anti-STAT6 (Cell Signaling). Secondary Abs: anti-rabbit IgG HRP-linked Ab (Cell Signaling), HRP labeled goat anti-mouse IgG (KPL), goat anti-rat IgG HRP-conjugated (Calbiochem), donkey anti-goat IgG HRP-conjugated (Santa Cruz).

Using a homogenizer (Ultra-Turrax IKA), pieces of whole colon tissues were lysed in cold RIPA lysis buffer (Sigma) containing protease inhibitors (Thermo Scientific), Na₃VO₄ (0.2 mM), NaF (20 mM) with incubation on ice for 15 min and intermittent vortexing. Total protein quantification of the samples was performed with BCA assay kit (Pierce). 20 μg aliquots of protein were separated by electrophoresis in 12% SDS PAGE gels (Bio-Rad) followed by transfer onto a PVDF membrane (Roche). Membranes were blocked for 1 hour, followed by incubating the membrane with primary antibody. Following primary antibodies were used: anti-p65 (Santa Cruz Biotechnology), anti-p105/p50 (eBioscience), anti-LMP7 and anti-LMP2 (Cell Signaling and Santa Cruz Biotechnology, respectively), anti-p-ERK (Cell Signaling) and anti-IRF4 (Santa Cruz Biotechnology). The analysed proteins were detected by chemiluminescence (Biostep) using ImmunoCruz (Santa Cruz Biotechnology). For detection of 20S proteasome subunits, the quantitative two-colour fluorescent immunoblot analysis was performed as described previously [16 (link)].

Chromatin was prepared according to the ChIP-IT Express Enzymatic protocol (Active Motif, Carlsbad, CA, USA). Immunoprecipitation was performed overnight using 2 μg of anti-NFATc1, anti-NFATc2, anti-IRF4 (Santa Cruz Biotechnology, Dallas, TX, USA), and anti-RNA Pol II and anti-IgG (Active Motif). Following incubation with antibody, DNA was eluted according to the Active Motif protocol and DNA was purified using the QIAquick PCR purification kit (Qiagen). Real-time qPCR was performed using iQSYBR green supermix (Bio-Rad) with the following primers: APOBEC3G ChIP-F 5′-GGG GAG GGG CTT GTG C-3′ and APOBEC3G ChIP-R 5′-AAG GCA ATT GCA AAG GGA A-3′. PCR was performed in triplicate with reaction conditions of 95 °C for 10 min followed by 40 cycles of 95 °C for 30 s, 60 °C for 1 min. Fold enrichment was calculated for each ChIP antibody used as quantity of ChIP DNA divided by amount of IgG DNA.