Nupage bis tris protein gel

Protein samples were resolved via SDS-PAGE using 10% (w/v) or 4–12% (w/v) gradient NuPAGE™ Bis–Tris Protein Gels (Life Technologies Australia Pty Ltd) and stained using Simply Safe Stain (Invitrogen) according to the manufacturer protocol. To conduct western immunoassay proteins in SDS-PAGE gels were transferred to a polyvinylidene difluoride membrane (Life Technologies) using Bolt™ Transfer Buffer (Life Technologies). Membranes were blocked using 5% (w/v) skimmed milk powder in PBS buffer (137 mM NaCl, 10 mM NaH₂PO₄·H₂O, 2.7 mM KCl, pH 7.4) for one hour. The anti-His-tag monoclonal antibody (Sigma-Aldrich) was diluted in PBST buffer (137 mM NaCl, 10 mM NaH₂PO₄, 2.7 mM KCl, with 0.2% Tween-20, pH 7.4) and used to probe the membrane overnight at 4º C in the same blocking solution. The blot was washed four times in 25 mL of PBST buffer for 10 min each then incubated at room temperature for 1 h with rabbit anti-mouse IgG horseradish peroxidase (HRP)-conjugated antibody (Sigma-Aldrich) in blocking solution. Following three washes in PBST for 15 min each, HRP activity was detected using Immobilon™ Western HRP chemiluminescent substrate with a luminol peroxidase solution according to the manufacturer’s instructions (Millipore Corporation, MA, USA) with image capture using a LAS 3000 Imager (Fujifilm, Japan).

Both types of cells were lysed on ice with M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, USA) supplemented with 10% protease inhibitor cocktail (Roche, Mannheim, Germany) to isolate total protein and then measured by a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, USA) in accordance with the manufacturer’s instructions. Protein samples supplemented with loading buffer in equal proportions were electrophoresed on NuPAGE™ Bis-Tris Protein Gels (Invitrogen, Waltham, MA, USA) and then transferred onto polyvinylidene fluoride (PVDF) membranes. After incubation with 5% skim milk (5% w/v) for 2 h at room temperature, the membranes were co-incubated overnight at 4 °C with the following primary antibodies specific for GAPDH (1:1000; Cell Signaling Technology, USA), Runx2 (1:1000; Cell Signaling Technology, Danvers, MA, USA), Osx (1:1000; Abcam, Cambridge, UK), Ocn (1:2000; Abcam, UK), ELK4 (1:1000; Proteintech, Rosemont, IL, USA), GM130 (1:1000; Proteintech, USA), CD9 (1:1000; Proteintech, USA), and TSG101 (1:1000; Proteintech, USA). Next, the peroxidase-conjugated secondary antibody (1:5000; ZS-GB-BIO, Beijing, China) was utilized to treat the PVDF membranes for 2 h at room temperature. The ECL kit (Thermo Fisher Scientific, USA) was employed to visualize the signals. Densitometry analysis was conducted with ImageJ software.

Reagent grade (or better) Tris Base (2-amino-2-(hydroxymethyl)-1,3-propanediol), calcium chloride, hydrochloric acid, dithiothreitol (DTT), imidazole, isopropyl-β-D-thiogalactopyranoside (IPTG), Nα-benzoyl-DL-arginine-4-nitroanilide (BApNA), kanamycin, Luria Broth, and Terrific Broth were purchased from Fisher Scientific (Thermo Fisher Scientific, Waltham, MA). To visualize protease expression of bacterial lysates and of concentrated purified proteases, 4–12% (gradient) or 12% NuPAGE™ Bis-Tris Protein Gels (Invitrogen, Carlsbad, CA) were used and stained with either InVision™ His-Tag In-Gel Stain (Invitrogen #LC6030), SimplyBlue™ Safe Stain (Invitrogen #LC6065) or both. For western blots, AaET-specific and other custom antibodies were purchased from GenScript (Piscataway, NJ) and have been previously described [1 (link), 15 (link)].

Immunoblotting was performed as described in the previous study^{29 (link)}. Briefly, cells were washed once with PBS and lysed with modified RIPA buffer (10 mM Tris–HCl [pH 7.4], 150 mM NaCl, 1 mM EGTA, 1% NP-40, 0.25% sodium deoxycholate, 0.1% SDS) containing phosphatase (1 mM NaF and 1 mM Na₃VO₄) and protease inhibitors. Proteins were extracted on ice with vortexing three times for 30 min and lysates were centrifugated at 10,000×g for 10 min at 4 °C. The supernatants were used for immunoblotting after boiling in 1 × SDS-sample loading buffer with reducing agent at 97 °C for 7 min. Protein concentration was analyzed by the BCA method (Sigma). Each protein sample (20–40 μg) was electrophoresed on 4–12% NuPAGE Bis–Tris Protein gels (Invitrogen) at a constant current of 20 mA, followed by transfer to NC (nitrocellulose) membranes (GE Healthcare). A membrane of protein blots from various human normal tissues (TB37-Set-1) was purchased from G-Biosciences. Each membrane was blocked with 5% skim milk, followed by immunoblotting with the indicated antibodies. The blots were developed using chemiluminescence (Thermo Scientific), and the images were analyzed using the ImageJ software.

The total protein was extracted from MC3T3-E1 cells with M-PER Mammalian Protein Extraction Reagent (Thermo Scientific, USA) including a 10% protease inhibitor cocktail (Roche, Switzerland). The protein concentration was measured by a Pierce^™ BCA Protein Assay Kit (Thermo Scientific, USA). Equal amounts of protein samples containing loading buffer were subjected to electrophoresis on NuPAGE^™ Bis–Tris Protein Gels (Invitrogen, USA) for 2 h. Proteins were transferred to polyvinylidene difluoride membranes, and the membranes were blocked in 5% skim milk for 4 h. Then, the PVDF membranes were incubated with primary antibodies specific for Runx2 (1:1000, Cell Signaling Technology, USA), Caspase-3 (1:1000, Cell Signaling Technology, USA), and β-actin (1:1000, Cell Signaling Technology, USA) overnight at 4 °C. The next day, after washing with TBST, the PVDF membranes were incubated with peroxidase-conjugated secondary antibodies (1:5000, ZSGB-BIO, China) for 1 h. The signals were visualized by Super Signal West substrate (Thermo Fisher Scientific, USA). Densitometric analyses of the bands were performed using Tanon Imaging software.

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).