Odyssey image station

Triton X-100 lysis buffer (containing protease- and phosphatase-inhibitors) was used to lyse lung tissue or cells, as previously described (Catravas et al., 2010 (link)). Then, the samples were centrifuged at 20,000 g at 4°C for 20 min and the supernatant was used to calculate the protein concentration by the BCA Protein Assay (Thermo Fisher, Waltham, MA). Tissue and cell extracts (17 μg) were separated using 4–20% Tris-SDS-Glycine PAGE, transferred to Immuno-Blot PVDF membrane by electrophoresis (Bio-Rad Laboratories, Hercules, CA), and then blocked in a Tris-buffered saline solution containing 5% nonfat milk. The membranes were probed with antibodies against SOX18 mouse (Santa Cruz, Dallas, TX) and rabbit (Thermo Fisher, Waltham, MA), Claudin5 mouse (Thermo Fisher, Waltham, MA), pNF-κB pS536 rabbit (Cell Signaling, Danvers, MA), NF-κB rabbit (Cell Signaling, Danvers, MA) and the corresponding secondary antibodies against rabbit and mouse (Thermo Fisher, Waltham, MA). Protein expression was normalized by re-probing with anti-β-actin (Sigma Aldrich, Burlington, MA). Reactive bands were visualized using chemiluminescence (Super Signal West Femto; Pierce, Rockford, IL) on a LI-COR Odyssey image station (Lincoln, NE). Bands were quantified using LI-COR Image Station software.

Brain tissue or cells were lysed with lysis buffer and subjected to a 15,000 g spin to remove nuclei and debris. Total protein concentrations were determined using the Enhanced BCA Protein Assay Reagent (Beyotime). Equal amounts (30 μg protein) were loaded onto SDS‐PAGE gels. Proteins were transferred onto polyvinylidene flouride (PVDF) membranes. Membranes were blocked with 5% dry milk solution for 1 hr and then incubated with primary antibodies overnight at 4°C. Membranes were washed with TBST, and protein bands were visualized using horseradish peroxidase‐conjugated species‐specific secondary antibodies. GAPDH was used as loading control. Images were captured, and band intensities were quantified using an Odyssey Image Station (LI‐COR).

Total cell lysates were prepared using radioimmunoprecipitation assay buffer (RIPA) or 2x Laemmli buffer supplemented with proteinase inhibitor cocktail (Sigma, USA) and phosphatase inhibitor cocktail (Sigma,USA). Liver samples were lysed and sonicated in T-PER (Thermo Fisher Scientific, Rockford, USA) per manufacturer’s protocol. Protein concentrations were determined using abicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Rockford, USA). Protein levels were analyzed by Western blot, as previously described^{31 (link),32 (link)}. β-Actin or GAPDH levels were also determined as controls. Protein bands were detected by chemiluminescence (Kodak Digital Science Image Station) or infrared fluorescence (Odyssey Image station and Image Studio, LI-COR Biosciences, Lincoln, NE, USA).

Brain tissue or cells were lysed with lysis buffer and subjected to a 12,000 rpm centrifugation. Total protein was determined using the BCA Protein Assay Reagent (ThermoFisher, USA). Fifty micrograms of protein and sample buffer was loaded onto 10% SDS-PAGE gels, and then the gels were transferred to PVDF membranes. Membranes were blocked and then incubated with primary antibodies anti-RIPK1 antibody (1:1,000), anti-RIPK3(1:1,000), anti-MLKL (1:1,000), or anti-pMLKL antibody (1:500) overnight at 4 °C. After washing three times with TBST, membranes were incubated with secondary peroxidase-conjugated antibodies, and protein blots were visualized using the ECL kit. GAPDH was used as a loading control. Images were captured, and band intensities were quantified using an Odyssey Image Station (LI-COR, USA).

Proteins were extracted by cell lysis (RIPA buffer supplemented with cOmplete [Roche, 11697498001]) 48 h post-transfection, loaded at 50 μg per well (Laemmli Sample Buffer) on 10% polyacrylamide gel, and transferred to polyvinylidene difluoride (Bio-Rad, Immun-Blot LF PVDF) for 1 h at 100 V in 4 °C. Blots were blocked for 1 h at 4 °C with Intercept Blocking Buffer (Li-Cor, 927-66003) and immunoblotted with primary antibodies overnight at 4 °C, and secondary antibodies for 1 h at RT. Antibodies: rabbit anti-CLTA (Proteintech, 10852-1-AP; 1:1000), mouse anti-GAPDH (Cell Signaling Technology, 97166; 1:1000), goat anti-mouse IgG (Li-Cor, 925-68020, IRDye 680LT; 1:20,000) and goat anti-rabbit IgG (Li-Cor, 925-32211, IRDye 800CW; 1:15,000). All antibodies were diluted in Intercept Antibody Diluent (Li-Cor, 927-66003). Western blots were visualized using an Odyssey Image Station (Li-Cor) and the Odyssey Application Software (3.0, Li-Cor).