Rabbit anti p65

Hela shCnt and HeLa shMyD88 cells were activated with 20 ng/mL IL-1β or 10 ng/mL TNFα (ProSpec, Rehovot, Israel) for 30 min or 1 h, respectively. Cells were then fixed (3.7% PFA in PBS for 10 min), permeabilized (0.25% Triton-X100) and blocked (2% BSA in TBS) at 4°C for 16 h. Cells were then stained with 0.6 µg/mL rabbit anti-p65 in 2% BSA in TBS (Santa Cruz Biotechnology, Dallas, TX, USA) followed by 0.5 µg/mL CY-3 goat anti-rabbit antibody (Jackson ImmunoResearch, Baltimore Pike, PA, USA). Cytoplasmic vs. nuclear localization was analyzed by fluorescent microscopy (15 (link)) (Nikon-Ti microscope).

Protocols for immunoblotting analysis have been described in detail previously [19 (link)]. Tissue lysates were prepared from the third mammary glands. Equal amounts of protein were separated by SDS-PAGE and transferred to nitrocellulose membrane. The membrane was probed with the primary antibody overnight at 4°C, and incubated with secondary antibodies conjugated with the HRP for 30min. Finally, the membrane was washed and scanned with an Odyssey Infrared Imaging System (LI-COR Biotechnology, Lincoln, NE, USA). The primary antibodies used in this study were mouse anti-tubulin(Sigma-Aldrich Co., St. Louis, MO, USA), rabbit anti-p50, rabbit anti-p65, rabbit anti-β-catenin, goat anti-lamin B and goat anti-β-actin (Santa-Cruz Biotechnology, Santa Cruz, CA, USA). Nuclear and cytosolic fractions were prepared by the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (PIERCE Biotechnology, Rockford, IL, USA) according to the manufacturer’s instructions. The protein levels were quantified by Quantity One software.

HEK293 cells were transiently transfected for 24 hr with HOIL-1, V5-His-HOIP, Sharpin, and/or UBE2L3. Cells were detached with TrypLE (Life Technologies), stimulated with TNF for 30 min, fixed with BD cytofix, permeabilized with 0.1% Triton X-100, and stained with rabbit anti-p65 (Santa Cruz), PE-conjugated anti-UBE2L3, and anti-V5-AlexaFluor647. Cells were washed, incubated with AlexaFluor488 F(ab)₂ donkey anti-rabbit IgG (Jackson), washed, stained with DAPI. 20,000 cells per condition were acquired on Imagestream X imaging flow cytometer (Amnis). For ex vivo cell analysis, PBMCs were isolated via Histopaque from blood samples from previously genotyped healthy individuals (TwinsUK). CD19⁺ B cells were isolated by magnetic bead positive selection (Miltenyi) and CD14⁺ monocytes were isolated by negative selection (Miltenyi). Endotoxin-free MACS buffer was used throughout. Cell purity was confirmed by flow cytometry. B cells and monocytes were cultured in RPMI and stimulated with 0.1 μg/ml CD40L (Enzo) and 10 ng/ml TNF (Axxora), respectively, for up to 60 min. Cells were fixed and stained for p65 as above, washed, and stained with DRAQ5 (eBioscience), and 15,000–20,000 events per sample were acquired by Imagestream X. Data analysis was entirely automated with IDEAS software batch function applied to the entire cohort and performed fully blinded to genotype.

Western blot analysis was performed as described to analyze the expression of p65, c-Rel, Bcl-xL, Bax and β-Actin in neurons from each group [46 (link)]. Cell extracts were collected on ice in a RIPA lysis buffer (Beyotime, China) and then centrifuged at 14000×g at 4°C for 10 min. Proteins were separated on 12% SDS-polyacrylamide gels and then electrotransferred onto a PVDF membrane (Millipore, USA). The membranes were blocked in 5% non-fat milk in TBS and probed with primary antibodies to rabbit anti-p65 (1:500, Santa Cruz, USA), rabbit anti-c-Rel (1:500, Santa Cruz, USA), rabbit anti-Bcl-xL (1:1000, Abcam, UK), rabbit anti-Bax (1:1000, Abcam, UK), rabbit anti-β-Actin (1:1000, Abcam, UK) and then incubated with a goat anti-rabbit IgG-HRP secondary antibody (1:8000, Abcam, UK). The optical densities of the antibody-specific bands were detected with ECL reagent kits and analyzed with Image J software (1.46r).

Primary antibodies used were rabbit anti‐p65 from Santa Cruz (clone C‐20, ref. sc‐372) at 1/250, mouse anti‐myc9E10 (developed by Bishop, J.M. was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at the University of Iowa), mouse anti‐HA (Eurogentec, clone 16B12, ref. MMS‐101R), rabbit anti‐HA (Sigma, ref. H6908), rabbit anti‐GFP (Amsbio, ref. TP401), rabbit anti‐UBAP1 (Proteintech, ref. 12385‐1‐AP), mouse anti‐His (Sigma, clone HIS‐1, ref. H1029), mouse anti‐FLAG (Sigma, clone M2, ref. F1804) all at 1/1000 and mouse anti‐TNFR1 (Santa Cruz, clone H‐5, ref. sc‐8436) and rabbit anti‐TSG101 (Atlas Antibodies, ref. HPA006161) both at 1/200. Rabbit polyclonal anti‐PumA serum was obtained by repeated immunization of rabbits with purified PumA (Eurogentec) and was used at 1/1,000 for Western blot and for immunofluorescence microscopy. Purified BtpA was used to obtain chicken anti‐BtpA (Eurogentec). Anti‐EF‐Tu antibody (kind gift from R. Voulhoux) was used at 1/10,000.
Secondary antibodies used were anti‐rabbit, mouse, chicken or rat conjugated with Alexas‐488, ‐555 or ‐647 all from Jackson ImmunoResearch. When necessary, phalloidin‐568 (1/1,000) was used to label the actin cytoskeleton and DAPI nuclear dye (1/1,000) for the host cell nucleus. For Western blots, anti‐mouse or rabbit‐HRP antibodies were used at 1/5,000.