Anti irf5

Lipopolysaccharide (Escherichia coli 0111:B4) was purchased from Sigma. CL264 and Poly I:C were purchased from InvivoGen. Murine macrophage colony-stimulating factor (M-CSF) was obtained from PeproTech. Anti-Blimp-1 antibody, anti-IRF5, anti-TRAF2 antibodies were obtained from cell signaling technology. Anti-β-actin antibody was obtained from Santa Cruz.

Plasmids encoding Myc-tagged IRF5 isoforms were a kind gift of Dr. Frank Neipel (Virologisches Institut - Klinische und Molekulare Virologie, Erlangen, Germany). Plasmids encoding Xpress-TRIM21 and GST-TRIM21 PRY/SPRY domain were a gift from Dr. David Rhodes (Cambridge Institute for Medical Research, Cambridge, UK). HA-ubiquitin wild type and mutants were a gift from Dr. James Burrows (Centre for Cancer Research and Cell Biology, Belfast, UK). Plasmids encoding FLAG-tagged IRF5 full length and deletion mutants were described previously [24] (link). Myc-MyD88 construct was a kind gift from Dr. Alberto Mantovani (Istituto Clinico Humanitas, Milan, Italy). pGL3-IFNA4 luciferase and pGL4-TK-Renilla were a kind gift from Dr. John Hiscott (Lady Davis Institute, Montreal, Canada) and Dr. Kate Fitzgerald (UMASS, Massachusetts, USA), respectively. Plasmids encoding shRNA targeting TRIM21 and scrambled negative control were described previously [16] (link). TLR ligands were purchased from InvivoGen (California, USA). Primary antibodies used were anti-FLAG (Sigma), anti-c-Myc and anti-β-Actin (Abcam), anti-GST (GE Healthcare), anti-Xpress (Invitrogen), anti-IRF5 (Cell Signaling) and anti-α-actinin, anti-HA and anti-TRIM21 (Santa Cruz).

Fresh isolated or treated DCs were rinsed with cold PBS and lysed in lysis buffer (Beyotime Biotechnology, Shanghai, China). The protein concentration was measured using NanoDrop. Equal amounts of protein were separated by 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes; nonspecific sites were blocked with 5% BSA in TBST and the membranes were then incubated with dilutions of the primary antibody as recommended by the manufacturer. The antibodies used are as follows: anti-IRF5(1:1000, 96527), anti-β actin (1:5000, 3700) (CellSignaling, Beverly, MA, USA), HRP conjugated anti-rabbit secondary antibody (1:2000, Invitrogen, Grand Island, NY, USA). Western blots were visualized using the enhanced chemiluminescent (ECL) reagent (Thermo Fisher).

BMDMs were lysed with 1× sample buffer (Tris [pH 8], 2% SDS, 10 mM EDTA, 0.01% bromophenol blue, 25 mM 1,4-dithio-DL-threitol, and 250 mM 2-ME), heated to 95°C for 10 min and separated by 10% sodium dodecyl sulphate–PAGE. Proteins were then transferred to 0.45-μm PVDF membranes (Millipore, Billerica, Massachusetts, USA), and membranes were washed and blocked with 5% skim milk in PBS. Antibodies were incubated overnight at 4°C in 5% BSA 0.05% Tween-20, and 0.1% sodium azide PBS. Membranes were washed three times (10 min) before addition of secondary HRP-coupled antibody in 5% skim milk PBS for 1 h at room temperature. PICOlucent (Gbioscience, St Louis, MO, USA) was then added, and the film (Agfa, Septestraat, Mortsel, Belgium) was exposed and developed. Anti-phospho IκBα, anti-IκBα, anti-phospho p65, anti-p65, anti-IRF5 and anti-actin antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA) and were used at 1/2000 dilutions. Anti-IRF4 antibody was obtained from Santa Cruz Biotechnology. The secondary polyclonal anti-mouse or -rabbit IgG HRP-coupled antibody was used at a 1/5000 dilution (Molecular probe, Eugene, Oregon, USA). Bands were quantitated and normalized with actin using ImageJ software.