Clarity max ecl substrate

The reactivity of pools of purified IgGs before immunization (n = 10) and after immunization (n = 10), or those depleted (n = 6), was assessed in a Western blot on sporozoite lysates. To generate the lysates, 1 million NF54 sporozoites were incubated with 100 μL of lysis buffer (150 mM Nacl, 20 mM Tris-Hcl, 1% triton, 1 mM EDTA at pH 7.5 and 1× protease inhibition cocktail; Thermo Fisher Scientific) for 15 minutes on ice followed by a 10-minute centrifugation at 13,000g at 4°C. Protein lysate corresponding to 1 × 10⁵ sporozoites were loaded per well on a 4%–12% Bis-Tris Protein Gels. Proteins were transferred into a nitrocellulose membrane (Bio-Rad), and strips were made. After blocking for 1 h with 5% milk in PBST, the blots were incubated for 3 hours with 5 μg/mL of CIS43 (CSP-specific mAb; ref. 42 (link)) or pre- or postimmunization total or depleted purified IgGs tested at 5-point 1:2 dilution at a maximum concentration of 20 μg/mL. After 3 washes with PBST, we incubated samples with secondary antibody (goat anti–human IgG [H+L], HRP, 1:30,000, Thermo Fisher Scientific catalog A18805; polyclonal). Then, blots were washed 6 times with PBST and incubated with Clarity max ECL substrate (Bio-Rad). The imaging was performed in ImageQuant LAS4000 (Bio-Rad). The intensity of the bands was analyzed using ImageJ (NIH).

Briefly, 60 μg of NF and Ty-82 cell lysate was spotted onto the nitrocellulose membrane at the center of the grid of a Bio-Dot SF Microfiltration Apparatus (BioRad, Milan, Italy). FN was used as a positive control. The membranes were dried and blocked by soaking in 5% BSA in TBS-T for 1 h at room temperature. The membranes were incubated for 1 h at room temperature with monoclonal antibodies anti-fibronectin BC-1 (1:500, Abcam, Cambridge, UK), L19-sip (1 µg/mL, Philogen SpA, Siena, Italy), and anti-β-Actin (Cell Signaling Technology, Danvers, MA, USA, 1:1000) diluted in BSA/TBS-T. After three washings with TBS-T, the membranes were incubated with secondary antibody HRP-conjugated goat anti-mouse IgG (BioRad, Milan, Italy, 1:1000) for 30 min at RT. Subsequently, the membranes were washed three times with TBS-T to remove unbound antibody and then incubated with Clarity Max ECL Substrate (BioRad, Milan, Italy) for 1 min. Chemiluminescence was detected with ChemiDoc MP Imaging System.

Total of mouse splenocytes, CD11b⁺ cells, BMDM, or monocytes from patients were lysed with RIPA buffer solution with a protease inhibitor cocktail. After 15 min on ice, lysates were centrifuged at 13,000 × g for 10 min at 4 °C. The proteins were separated in a 12%-acrylamide (caspase-11 and caspase-8) or 4–12% acrylamide (caspase-1/caspase-4/GSDM-D) NUPAGE Bis-tris Protein gels (Invitrogen) and transferred onto nitrocellulose membranes. The membranes were incubated with caspase-11 (Novus Biologicals, 1:1000), caspase-1 (Adipogen, 1:1000), caspase-4 (Cell Signaling, 1:1000), anti-mouse and anti-human caspase-8 (Enzo Life Science, 1:1000), anti-mouse cleaved caspase-8 (Cell Signaling, 1:500), anti-GSDM-D (sigma, 1:1000) and β-actin (Sigma, 1:2000) specific antibodies, then incubated with HRP-conjugated secondary antibodies (Jackson ImmunoResearch, 1:25,000) and detected with Clarity Max ECL Substrate (Biorad) using ImageLab Touch Software V6.0.1 (Bio-Rad). Bands quantification was performed with ImageStudio V5.2. Uncropped western blot is available in the Supplementary Information file (Supplementary Figs. 5–11).

Freshly isolated keratinocyte populations were harvested for CD271 in lysis buffer pH 7.5 (150 mM NaCl, 15 mMMgCl, 1 mM EGTA, 50 mM Hepes, 10% Glycerol, 1% Triton), or in RIPA buffer. WB was performed as previously described.^{9 (link)} Membranes were first incubated in blocking buffer and then overnight at 4 °C with primary antibodies: mouse anti-human CD271, clone ME20.4 (Upstate, Lake Placid, NY, USA), mouse anti-human β1 integrin, clone 12G10 (Abcam, Cambridge, UK), rabbit anti-FOXM1, clone D3F2B (Cell Signaling Tech, Danvers, MA, USA), rabbit anti-p63(ΔN) (BioLegend, San Diego, CA, USA), rabbit anti-Survivin (Boster, Pleasanton, CA, USA), rabbit anti-p16INK4 (Bethyl Laboratories Inc, Montgomery, TX, USA), rabbit anti-Ki67 (Abcam), mouse anti-human β-actin, clone AC-15 (Sigma). Then membranes were incubated with anti-mouse or anti-rabbit peroxidase-conjugated secondary antibodies (Biorad, Hercules, CA). Membranes were developed in Clarity Max ECL substrate (BioRad) and images were captured with ChemiDoc Imaging System (Biorad). The band intensity was quantitatively determined using Fiji-ImageJ software (Wayne Rasband, National Institute of Mental Health, Bethesda, MD, USA), and protein levels’ intensity was normalized to β-actin expression.