Anti irf1 antibody

Cells were lysed in 100 μl of Frackelton lysis buffer (10 mM Tris [pH 7.4], 50 mM NaCl, 30 mM Na₄P₂O₇, 50 mM NaF, 2 mM EDTA, 1% Triton X-100, 1 mM DTT, 1 mM PMSF, and 1X protease inhibitor cocktail). Cells were incubated on a rotating wheel at 4°C for 30 min and subsequently centrifuged at 20,000 × g at 4°C for 30 min. The supernatant was transferred to a new tube and 10 μl (10% of the lysate used for the immunoprecipitations [IPs]) was collected as input. 300 μg of lysates were incubated overnight at 4°C on a rotating wheel with an IgG Isotype Control (Cell Signaling Technology, 1:300), anti-Ollas (Novus, 1:100), or anti-IRF1 antibody (Cell Signaling Technology, 1:100). The next day, magnetic beads (Pierce Protein A/G Magnetic Beads, Thermo Fisher Scientific, 88803) used for anti-IRF1 antibody IPs, or Protein G Sepharose beads (Protein G Sepharose 4 Fast Flow, Sigma-Aldrich, GE17-0618-01) used for anti-Ollas antibody IPs, were blocked by rotation in 3% BSA in Frackelton Buffer for 1 hr at 4°C. 25 μl of beads were added to 300 μg of lysates and rotated for 2 hr at 4°C. Then, the beads were washed five times with 1 ml of RIPA buffer, supplemented with 300 mM NaCl. Proteins were eluted by boiling in 2X disruption buffer (2.1 M urea, 667 mM β-mercaptoethanol, and 1.4% SDS) for 5 min at 95°C.

200,000 HeLa cells/35 mm well were seeded onto coverslips (Marienfeld, 630-2190) and transfected using polyethylenimine (PEI) transfection reagent (Polysciences, 23966) in a 1:6 (wt/wt) DNA/PEI ratio in non-supplemented DMEM with lentiviral plasmids expressing MYC-tagged wtIRF1 and FLAG-tagged wtSPOP. 48 hr after transfection, cells were fixed with 4% paraformaldehyde (PFA) for 15 min. Similarly, 250,000 RKO cells stably expressing HA-tagged wtIRF1 and FLAG-tagged wtSPOP were seeded onto coverslips and after 48 hr stimulated with epoxomicin (10 µM, 4 hr) followed by fixation. Cells were permeabilized with 0.25% Triton X-100 in PBS for 5 min, followed by blocking of non-specific sites by incubation with 1% BSA for 30 min at RT. coverslips were incubated for 1 hr at RT with primary anti-IRF1 antibody (Cell Signaling Technology, 1:100), anti-HA (Cell Signaling Technology, 1:1000), or anti-FLAG (M2, 1:500) antibody in 1% BSA, followed by incubation with anti-rabbit IgG Alexa Fluor 488 (Abcam, 1:800), anti-mouse Alexa Fluor 647 (Invitrogen, 1:1000), or anti-mouse IgG DyLight 550 (Invitrogen, 1:500) secondary antibodies, and incubation for 5 min with 0.4X Hoechst (Thermo Fisher Scientific, H3569) in PBS. The coverslips were mounted using ProLong Gold Antifade Mountant (Invitrogen, P36934). Images were collected using a Zeiss LSM 980 confocal microscope at ×40 magnification.