Clarity ecl kit

Cells obtained from BAL of WT or Ripk3^-/- mice at day 3 post-infection or BMD-Mφ were lysed in lysis buffer (1% Triton X-100, 150mM NaCl, 20mM Hepes pH7.5, 10% glycerol, 1mM EDTA, supplemented with anti-protease and anti-phosphatase cocktails, Roche) and protein concentration was determined using BCA assay (Pierce). 20 μg of protein were resolved by SDS-PAGE and transferred onto PVDF membranes (Biorad). Membranes were blocked and incubated overnight at 4°C with gentle agitation with primary antibodies. The following primary antibodies were used: anti-RIPK3 (1:1000, Proscience #2283), anti-phospho-PKR (1:200, Santa Cruz Biotechnology #sc-101784), anti-PKR (1:200, Santa Cruz Biotechnology #sc-1702), anti-RIPK1 (1:1000, Cell Signaling Technology #3493), anti-phospho-IRF3 (1:500, CST #4947), anti-IRF3 (1:1000, CST #4302), anti-phospho-eIF2α (1:1000, CST #3597), anti-eIF2α (1:1000, CST #5324), anti-MAVS (1:1000, CST #4983), anti-TBK1 (1:1000, CST #3504), anti-pTBK1 (1:1000, CST #13498), anti-CYPD (1:1000, Calbiochem #AP1035) and anti-actin (1:10000, Sigma-Aldrich #2066). Primary antibodies were followed by HRP-conjugated secondary antibodies and signal was detected using Clarity ECL kit (Biorad) and acquired on Chemidoc MP System (Biorad). Densitometry analyses were performed using ImageJ software (NIH).

Cytosolic and nuclear extracts were prepared using a Nuclear/Cytosol Fractionation Kit (BioVision Research, USA). Cytosolic extracts (for NF-κBp65, NF-κBp50, COX-2, iNOS, STAT3, c-Myc, Bcl-xl, Nrf2, SOD, GSTP, EGFR, Akt, p-Akt, Keap1, IKKα/β and β-actin) or nuclear extracts (for NF-κBp65, NF-κBp50, STAT3, p-STAT3, Nrf2, p-Nrf2 and lamin) were separated on 7.5%, 10% or 12% SDS–PAGE slab gels. Then, the proteins were transferred to a nitrocellulose Immobilon-P membrane. After blocking for 2 h with 10% skimmed milk, proteins were incubated with primary antibodies against NF-κBp65, NF-κBp50, COX-2, iNOS, STAT3, p-STAT3, c-Myc, Bcl-xl, Nrf2, p-Nrf2, SOD, GSTP, EGFR, Akt, p-Akt, Keap1, IKKα/β, β-actin, and lamin. Alkaline phosphatase (AP)-labeled anti-rabbit IgG, anti-goat IgG, and anti-mouse IgG secondary antibodies (Bio-Rad Laboratories, USA), as well as horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Boster Bio, USA) secondary antibodies were used in the staining reaction. Bands were visualized by the AP Conjugate Substrate Kit NBT/BCIP or the chemiluminescent HRP substrate of the Clarity ECL Kit (Bio–Rad Laboratories, USA). The amount of immunoreactive product in each lane was determined using a ChemiDoc Imaging System (Bio–Rad Laboratories, USA). Values were calculated as relative absorbance units (RQ) per mg of protein and expressed as a percentage of the control.

Proteins were run on Bis-Tris gradient acrylamide gels and transferred to nitrocellulose membranes using the iBlot system (Life Technologies). Membranes were incubated in blocking solution (50 mM Tris pH 7.5, 300 mM NaCl, 3% wt/vol dry milk powder, and 0.05% Tween-20) for 10–60 min, primary antibody overnight at 4°C, washed, incubated in HRP-conjugated secondary antibodies (Jackson Immuno) for 1 hr at room temperature and then detected using the Bio-Rad Clarity ECL kit on a Bio-Rad ChemiDoc XRS+ imaging system. To prepare hippocampal lysates, 100 mg of hippocampal tissue was homogenized in 1 ml of reducing sample buffer.

Cell pellets were resuspended in lysis buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100 (Sigma-Aldrich), protease/phosphatase inhibitors cocktail (Roche) and 20 nM N-ethylmaleimide (NEM) (Sigma-Aldrich) to prevent deSUMOylation [38 (link)], sonicated and incubated for 15 min on ice. BCA protein assay (Thermo Fisher Scientific) was used to quantify protein lysates and 30 μg protein samples were run on 10% NuPAGE Bis–Tris pre-cast polyacrylamide gels (Thermo Fisher Scientific) by SDS-PAGE and transferred to nitrocellulose membranes. Immunoblots were performed with specific primary antibodies (listed in Supplementary Table 1), diluted in 5% milk in TBS with 0.1% Tween-20 (Sigma-Aldrich). The HRP-conjugated secondary antibodies were detected by the Clarity ECL kit (Biorad), while the Veriblot reagent (Abcam, Cambridge, UK) was used to avoid interference of denatured IgG chains in immunoprecipation detection assays (see below). Densitometric analyses were performed using ImageJ software (NIH).