Supersignal femto substrate

For caspase‐1 immunoblotting, BMDMs and supernatant were lysed in lysis buffer and sample loading buffer containing SDS and 100 mM DTT. Proteins were separated on 8–12% polyacrylamide gels. Following electrophoretic transfer of proteins onto PVDF membranes (IPVH00010, Millipore), membranes were blocked in 5% skim milk in TBST and incubated overnight with primary antibodies against mouse caspase‐1 (1:1,000 dilution, AG‐20B‐0042, Adipogen), human caspase‐1 (1:1,000 dilution, 3866, Cell Signaling Technologies), mouse GSDMD (1:3,000 dilution, ab209845, Abcam), human GSDMD (1:1,000 dilution, ab215203, Abcam), NINJ1 (1:1,000 dilution, A16406, ABclonal), CD14 (1:1,000 dilution, 17000‐1‐AP, Proteintech), and GAPDH (1:10,000 dilution, 5174, Cell Signaling Technologies). PVDF membranes were then incubated with HRP‐conjugated secondary antibody (1:5,000) for 1 h and proteins were visualized using the Super Signal Femto substrate (34095, ThermoFisher Scientific) and the ChemiDoc™ Touch Imaging System (BioRad).

For Western blot analysis, cells were lysed in RIPA buffer and heated at 95 °C for 5 min with SDS sample buffer. Afterwards, proteins were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes by semi-dry electroblotting. Five percent milk in PBS containing 0.05% Tween (PBS-T) was used to block the membranes. Subsequently, membranes were probed with primary antibodies α-FLAG (1:1000, Sigma-Aldrich, Catalogue Number F3165), α-KRT23 (1:2000, Thermo Fisher, Catalogue Number PA5-50198), α-KRT23 (1:1000, Abcam, Catalogue Number ab156569) α-HCV-NS3 #337 mAb (1:1000), α-GAPDH (1:1000, Sigma-Aldrich, Catalogue Number G9545), and α-β-actin (1:20000, Sigma-Aldrich, Catalogue Number A3854) over night at 4 °C, followed by incubation with secondary horseradish peroxidase conjugated antibodies (Sigma-Aldrich) for 1 h at room temperature. For analysis, membranes were incubated with the ECL Plus detection system (GE Healthcare), SuperSignal Femto Substrate (Thermo Fisher), and Pierce™ ECL Plus Western Blotting Substrate (Thermo Fisher).

Western blotting was performed as previously described [9 (link)]. Briefly, the cells were lysed in an RIPA buffer containing protease inhibitors. The protein concentration was measured according to the manufacturer’s instructions (#23227, Thermo Fisher, Dreieich, Germany). The cell lysates were resolved on a 2–14% SDS-PAGE gel and transferred onto a nitrocellulose membrane. The membranes were blocked with 5% BSA in TBS + 0.05% Tween 20 for 1 h at room temperature. The primary antibodies were incubated overnight at 4 °C. JL-8 monoclonal mouse anti-GFP antibody (1:1000, #632380, Takara Bio, Saint-Germain-en-Laye, France) was used to detect a conserved amino acid sequence in GFP-derived fluorophores included in the RFP. An HRP-conjugated donkey anti-mouse antibody was used as a secondary antibody (1:10,000, #115-035-003, Jackson ImmunoResearch, Ely, UK). For loading control, an anti-actin-HRP antibody (1:10,000, #A3854, Sigma-Aldrich, Darmstadt, Germany) was used (incubation: 1 h at room temperature). An ECL reaction was performed using a SuperSignal Femto Substrate (#34095, Thermo Fisher, Dreieich, Germany).

For dot blot analysis, 3 µL of patient plasma were spotted on polyvinylidene difluoride membranes and air-dried for 1 h at room temperature. Membranes were blocked with 5% milk in PBS containing 0.05% Tween (PBS-T) for 1 h at room temperature and subsequently probed with primary antibodies (α-KRT23, 1:1000, kindly provided by Pavel Strnad [8 (link)]) over night at 4 °C, followed by incubation with secondary horseradish peroxidase conjugated antibodies (Sigma-Aldrich) for 1 h at room temperature. For analysis, membranes were incubated with the ECL Plus detection system (GE Healthcare) and SuperSignal Femto Substrate (Thermo Fisher). Fiji was used to calculate signal intensities of KRT23 on the different dot blots. Therefore, regions of interest with the same size were selected in all samples, and mean grey values were quantified.

Cell lysates were prepared in RIPA buffer with 1x protease and phosphatase inhibitor cocktails (RPI; Mount Prospect, IL), PUGNAC (50 μM; Sigma, St. Louis, MO), and Thiamet G (25 μM, Sigma) added to block removal of the O-GlcNAc modification and subjected to immunoblotting as previously described (24 (link)). Briefly, nitrocellulose membranes were probed with antisera for the following: (1) anti-mouse O-GlcNAc (1:1,000; clone CTD 110.6 Biolegend, San Diego, CA, USA), anti-rabbit OGT (1:5,000, Sigma), anti-rabbit OGA (1:5,000 Bethyl Laboratories, Montgomery, TX, USA), anti-rabbit phospho-PLCγ (8713S) (Cell Signaling, Danvers, MA, USA), anti-rabbit PLCγ (2822S) (Cell Signaling), anti-rabbit phospho-ERK (9101S) (Cell Signaling), anti-rabbit ERK (4695S) (Cell Signaling), and anti-mouse β-actin (Sigma). Probed blots were developed using enhanced chemiluminescence Supersignal Femto Substrate (Thermo Scientific; Grand Island, NY, USA). All blots were imaged using the GE Imaging System (GE Healthcare, USA) and densitometric analyses was performed using Image J (25 (link)).

The ipsilateral hemisphere of the cerebrum was homogenized with protease+phosphatase inhibitors (Roche, complete mini, DK Phosphosafe; Millipore), protein content quantified, aliquoted, and stored at −22°C. Immunoblotting was optimized and performed with standard Western blot principles on the ipsilateral hemisphere encompassing the lesion site. Homogenates were reduced, heated, and loaded at 25–50 μg into precast polyacrylamide gels [12% or 4–12% (α-spectrin) (NuPAGE; Life Technologies, Naerum, Denmark)] and gels run in MES buffer and transferred to polyvinylidene difluoride membrane. Membranes were washed in tris-buffered saline (TBS), and blocked in 5% skim milk powder or bovine serum albumin + TBS-(0.01% Tween) for 1 h at room temperature. Primary antibodies were applied in appropriate blocking solution overnight at 4°C and are listed in detail in Table 1. Secondary antibodies were applied in appropriate blocking solution: Horseradish peroxidase-conjugated-conjugated anti-rabbit/anti-mouse (Dako, Glostrup, Denmark) at 1:2000 and 1:3000, respectively, for 1 h at room temperature. Membranes were incubated in SuperSignal Femto substrate (34095; Thermo Scientific) and exposed with a CCD camera (Bio-Rad Chemidoc XRS imager, Copenhagen, Denmark). Images were quantified with ImageJ and reported relative to housekeeping protein, glyceraldehyde 3-phosphate dehydrogenase (GAPDH).