Anti irf5

ChIP assays were performed with SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology Japan, Tokyo, Japan) with anti-IRF1 (Santa Cruz Biotechnology), anti-IRF3(Santa Cruz Biotechnology), anti-IRF5 (Abcam), anti-IRF7 (Santa Cruz Biotechnology), anti-IRF8 (Santa Cruz Biotechnology), anti-IRF9 (Santa Cruz Biotechnology), normal goat IgG (Abcam) and normal rabbit IgG (Cell Signaling Technology) antibodies. ChIP signals were quantified by quantitative PCR analysis with a 7500 real-time PCR system (Applied Biosystems). Values obtained for immunoprecipitated samples (percent (%) input DNA) were normalized to values for respective normal IgG. The specific primer pairs for the Irf5 promoter region and P2rx4 promoter region, respectively, are described below.
Region 1: 5′-ATTTCTCAGGCCCTGTCTAAAGTG-3′ (forward),
5′-GGCACAGAGAGAGTTAGAGGAAGA-3′ (reverse)
Region 2: 5′-TATGGAGTCTTTCTGCACCCTGT-3′ (forward),
5′-TTCCAAGAACGAAGAGTCCCCTA-3′ (reverse)
ISRE-1: 5′-GCTGGCTCGTTTCAAGAATATT-3′ (forward),
5′-CGTACCCTGTAGCCGTCTATT-3′ (reverse)
ISRE-2: 5′-TCTACAGCCTGAAAGTCTATCATTG-3′ (forward),
5′-AAGGAATCTGAGAGGTACACACTG-3′ (reverse)
ISRE-3: 5′-GATAGGGAGAGGCTCGTTCA-3′ (forward),
5′-TAAAAGCTCGGGACCTGGAA-3′ (reverse)
ISRE-4: 5′-TACTGACCTGCCTCTTTTAAGGACA-3′ (forward),
5′-CGGAAAGAACTTTGAACCTTGAG-3′ (reverse)

A549 and THP-1 cells were treated with increasing concentrations of TSA (0, 1, 2.5, or 5 μM) or 0.1% DMSO (control). Western blot analysis was conducted as previously described [40 (link)]. The primary antibodies used were anti-GAPDH, anti-IRF5, and anti-Sp1 (Abcam) at dilutions of 1:200–3000.

Mouse CD43⁻ resting B cells, which were stimulated with 1 μg/ml anti-mouse IgM Ab and 100 μg/ml poly(I:C) for 72 h, were lysed in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate and 1% Nonidet P-40) in the presence of a protease inhibitor cocktail (Sigma-Aldrich). After 30 min incubation, the cells were sonicated by ultrasonication (Emerson Electric, St. Louis, MO, USA). Supernatants were collected after centrifugation at 15,000 g for 30 min and mixed with SDS sample buffer with 2-mercaptoethanol (Sigma-Aldrich). After 5 min boiling, the proteins were separated by NuPAGE 4–12% Bis-Tris gel electrophoresis (Invitrogen) and transferred onto polyvinylidene fluoride (PVDF) membrane (Merck Millipore). Anti-IRF4 (1:1,000 dilution, BioLegend), anti-IRF5 (1:1,000 dilution, Abcam, Cambridge, MA, USA), anti-IRF8 (1:1,000 dilution, Abcam), anti-ubiquitin (1:2,000 dilution, BioLegend), and anti-β-Actin antibodies (1:4,000 dilution, BioLegend) were used to detect the specific proteins. Chemiluminescence was developed using ECL Select Western Blotting Detection Reagent (GE Healthcare Life Sciences, Pittsburgh, PA, USA) and exposed to the LAS-3000 Mini Imaging System (FUJIFILM, Tokyo, Japan).

Intestinal tissues were harvested and lysed in RIPA lysis buffer containing protease inhibitors. An equal amount of protein from all the samples was separated by SDS-PAGE and then transferred to PVDF membranes. The following antibodies were applied: anti-IRF5 (1:1000, ab181553) from Abcam and anti-cleaved caspase-3 (1:1000, 9661S) and anti-GAPDH (1:3000, 3668S) from Cell Signaling Technology (Danvers, MA).