Supersignal west pico plus chemiluminescent

All cells were lysed in 1% NP40 buffer (150 mM NaCl, 50 mM Tris-HCl pH 8, 10% glycerol, 2 mM EDTA) supplemented with 1 mM cOmplete Mini protease inhibitors (Roche) and 1 mM sodium orthovanadate (ThermoFisher). Proteins were separated on a 4–20% SDS-PAGE gel (BioRad) and transferred to polyvinylidene difluoride (PVDF) membrane. Membrane was incubated in 5% BSA and 0.1% Tween-20 in PBS for 1 hr, incubated overnight at 4°C with primary antibodies (1:10000 anti-β-actin, Santa Cruz #sc-47778; 1:1000 anti-SHC, BD Transduction #610878; 1:1000 anti-ERK, Invitrogen #13–6200; 1:1000 anti-phospho-SHC (Tyr239/240), Cell Signaling #2434; 1:2000 anti-phospho-p44/42 ERK (Thr202/Tyr204), Cell Signaling #4370; 1:1000 anti-EGFR, Cell Signaling #2232; 1:700 anti-phospho-Thr/Tyr, Cell Signaling #9381; 1:500 anti-CLDN1, Invitrogen #37–4900; 1:350 anti-OCLN, Invitrogen #33–1500), then incubated for 1 hr at room temperature with horseradish peroxidase-conjugated secondary antibodies (1:10000 anti-rabbit, Cell Signaling #7074; 1:10000 anti-mouse, Cell Signaling #7076). Membrane was added SuperSignal West Pico PLUS Chemiluminescent or West Femto Maximum Sensitivity substrate (ThermoFisher) and exposed to CL-XPosure film (ThermoFisher).

The cells were harvested and lysed using RIPA buffer with protease inhibitor (Thermo Fisher Scientific), and the protein concentrations were determined by the BCA protein assay (Thermo Fisher Scientific). A total of 40 ug of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes as previously described [23 (link)]. Blots were incubated with primary antibodies against NLRP3 (#15101; Cell signaling technology, Danvers, MA, USA), ASC (#67824; Cell signaling technology), pro-IL-1β (sc-32294; 1:1,000 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), cleaved IL-1β (#83186; Cell signaling technology), pro-CASP1 (#3866; Cell signaling technology), cleaved CASP1 (#89332; Cell signaling technology), and GAPDH (sc-32233; Santa Cruz Biotechnology) at the 1:1,000 dilution, followed by an incubation with the secondary antibody conjugated to HRP for 1 h in room temperature. The bands were visualized using SuperSignal West Pico PLUS Chemiluminescent (Thermo Fisher Scientific) and luminescent image analyzer (ChemiDoc XRS + Systems, Bio-Rad Laboratories) at multiple exposure settings. For quantification, ImageJ software (National Institutes of Health) were used to analyzed the bands intensity.

Astrocyte cultures were washed twice with sterile 1X PBS and scraped from the dish with 100 μl RIPA buffer supplemented with one protease inhibitor cocktail tablet/10 ml (Roche 011836170001). The lysate was resuspended thoroughly and centrifuged at 15 000 RPM at 4°C for 10 min. The supernatant was collected, and the protein concentration was determined using the BCA method. The samples were mixed with loading buffer, and 30 μg of total protein was loaded on a NuPAGE 4–12% Bis-Tris protein gel (Invitrogen) and separated at 70 V. The proteins were dry-transferred into a nitrocellulose membrane. The membranes were blocked for 1 h in 5% dry milk powder reconstituted in TBS-T 0.01%; subsequently, total tau (DAKO A0024, 1:000) and β-actin (Santa Cruz sc4778, 1:5000) primary antibodies diluted in TBS-T 0.01% -BSA 3% were added, and the membranes were incubated ON at 4°C. The next day, the antibodies were replaced with the corresponding secondary HRP-conjugated antibodies (goat anti-mouse and goat anti-rabbit Jackson Immunoresearch 711035152 and 715035150, respectively) in 5% milk, and the membranes were incubated for 1 h at RT. According to the manufacturer's specification, the membranes were washed twice with TBS-T and visualized using SuperSignal West Pico PLUS Chemiluminescent (ThermoFisher) developing solution.