Western lightning plus ecl detection reagent

Serum IgG or IgA reactivity toward CBL or HhL was analyzed by immunoblot analysis. For screening multiple sera from Ctr and DC-LMP1/CD40 animals, the Mini-PROTEAN II Multiscreen Apparatus (Bio-Rad, Cat: 1704017) was used. CBL (30 µg per lane or 600 µg for Mini-PROTEAN II Multiscreen Apparatus) or HhL (20 µg per lane or 200 µg for Mini-PROTEAN II Multiscreen Apparatus) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Sera of mice were used as primary antibodies. Differences in serum antibody concentrations between Ctr and DC-LMP1/CD40 mice were adjusted by using 2.5 µg ml⁻¹ serum IgG or 1 µg ml⁻¹ serum IgA for all samples. In some experiments, mouse IgG1 anti-human heat shock protein 60 (aHSP60) antibody (clone LK-2, Enzo, Cat: ADI-SPA-807-E) was additionally used as the primary antibody (1:10,000 in PBS/1% nonfat dried milk). HRP-conjugated secondary antibodies were used as follows: goat anti-mouse IgG-HRP (SouthernBiotech, Cat: 1030–05; 1:10,000) or goat anti-mouse IgA-HRP (SouthernBiotech, Cat: 1040–05; 1:10,000). Western Lightning Plus-ECL Detection Reagent (PerkinElmer, Cat: NEL103E001EA) and X-ray films (Amersham, Cat: 45–001-504) were used for protein detection.

To obtain whole cell lysates, cells were extracted six days after transduction by use of Mammalian Protein Extraction Reagent (M-PER, Thermo Fisher Scientific) and subjected to at least three freeze-thaw cycles (− 80 °C/room temperature). Samples were run on a sodium-dodecyl sulfate/ 10% polyacrylamide gel and blotted onto a Westran S polyvinylidene difluoride membrane (GE Health Care). After blocking for 24 h in a 5% milk solution, proteins of interest were detected by incubation for 24 h with antibodies against MAP3K7, NF-κB p100/p52, NF-κB p105/p50, IκB (all Cell Signaling) used at a 1:1000 dilution in 5% milk/TBS-T (Tris-buffered saline with 0.5% Tween-20) or 5% BSA (bovine serum albumin)/TBS-T for phospho NF-κB p65 Ser536 (Cell Signaling). Incubation for 1 h with a peroxidase-conjugated anti-mouse or anti-rabbit antibody (both Sigma) was followed by signal detection with Western Lightning Plus-ECL detection reagent (PerkinElmer) using the Fusion FX detection system (Vilber Lourmat). ImageJ (1.48v) was used for quantification of results.

SK6 cells transfected with 3′NCR transcripts or HEK 293 cells transfected with flag-tagged plasmids expressing MDA5 or RIG-I were washed twice in ice-cold PBS and lysed at different times post-transfection (pt) in PBS containing 1% NP-40, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride and 1× Complete protease inhibitor cocktail (Roche, Basel, Switzerland). 20 μg of cell extracts was run on 10% SDS-PAGE gels, transferred onto nitrocellulose membrane and probed with the corresponding primary antibody. Blots were then incubated with the corresponding goat anti-mouse, goat anti-rabbit or rabbit anti-goat IgG (H + L) secondary antibody HRP conjugate (Thermo Scientific Pierce, Rockford, IL, USA). Protein bands were visualized using Western Lightning Plus-ECL detection reagents (Perkin Elmer Inc., Waltham, MA, USA) followed by exposure to X-ray film. Monoclonal anti-FLAG M2 (F1804) was purchased from Sigma. Mouse monoclonal anti-porcine Mx1 (AM39) was from Acris Antibodies. Goat polyclonal antibodies against human RIG-I (sc-48929) and MDA-5 (sc-48031) were purchased from Santa Cruz. Rabbit polyclonal antibody anti-βII tubulin has been previously described [29 (link)].