Cleaved gsdmd

Following colonic crypts homogenization, as described above, total protein was extracted by lysing tissue in ice-cold RIPA buffer supplemented with protease inhibitor cocktail (Santa Cruz #sc-24948), sodium orthovanadate, and PMSF. Protein concentrations were quantified using the BCA method. For each group, 15 μg of protein were transferred to a nitrocellulose membrane after electrophoretic separation. The membranes were blocked using 5% of either dry milk or BSA in Tris Buffered Saline + Tween20 (TBST) buffer. The nitrocellulose membrane was then incubated overnight with the following primary antibodies: cleaved GSDMD (Cell Signaling Technology, #50928), cleaved caspase-11 (Abcam, #ab180673), cleaved caspase-1 (Invitrogen #AB 5B10), and β-actin (BD Biosciences #612656) at 1:1000 dilution. After washing steps in TBST, the membrane was incubated at room temperature for 1 h with anti-rabbit IgG (H + L) or anti-mouse IgG (H + L) (both Jackson ImmunoResearch) at 1:15,000 dilution. Proteins were visualized using SuperSignalTM West Femto or SuperSignal West Pico PLUS (both Thermo Fisher) chemiluminescent detection kits.

Suspensions of hMSC-secreted exosomes were lysed in RIPA lysis buffer (Beyotime), and cell lysates were centrifuged at 10,000 g for 20 min. The supernatant was then collected for storage at − 80 °C. Suspensions of hMSC-secreted exosomes (16 μl) were loaded with 4 μl of loading buffer (5×, beyotime) at 100 °C water bath for 5 min, followed by centrifugation at 10,000×g 5 min at room temperature. Current for electrophoresis was 80 V for 30 min and then 120 V for 90 min. The membranes were transferred at 200 mA for 2 h. After membranes were washed in TBST, 5% fat-free milk was added for blocking for 1 h. The blot was probed with TBST-diluted primary antibodies of rabbit anti-rat β-actin (4970S, Cell Signaling), TSG101 (ab30871, Abcam), CD63 (ab217345, Abcam), Sirt1 (9475T, Cell Signaling), caspase-1 (24232S, Cell Signaling), cleaved-caspase-1 (89332S, Cell Signaling), GSDMD (93709S, Cell Signaling), cleaved-GSDMD (36425S, Cell Signaling), ASC (67824T, Cell Signaling), and NLRP3 (13158S, Cell Signaling) in a shaking bed for incubation for 1 h and subsequently probed with horse radish peroxidase-labeled goat anti-rabbit IgG secondary antibody (1:5000, Beijing ComWin Biotech Co., Ltd., Beijing, China) at room temperature for 1 h. After TBST wash for 3 × 10 min, the secondary antibody signals were detected.

Standard techniques were used for protein quantification, separation, transfer, and blotting in KLE and HEC-1A cells. Primary antibodies immunoblotting antibodies were used: ERRα (1: 500, ab76228, Abcam, London, UK), ERRα (1:500, E1G1J, Cell Signaling Technology, Massachusetts, USA), NLRP3 (1:500; 19,771-1AP, Proteintech, Wuhan, China), HIF-1α (1:500; 20,960-1AP, Proteintech, Wuhan, China), cleaved GSDMD (1:500; #36,425, Cell Signaling Technology, Massachusetts, USA), GSDMD (1:500, E9S1X, Cell Signaling Technology, Massachusetts, USA), GSDME (1:500, 84005S, Cell Signaling Technology, Massachusetts, USA), Caspase1 (22,915–1-AP, Proteintech, Wuhan, China), Caspase-1 (YT5743, Immunoway, USA), Caspase-1 (1:500, D7F10, Cell Signaling Technology, Massachusetts, USA), Caspase-3 (1:500, D3R6Y, Cell Signaling Technology, Massachusetts, USA), IL-18 (1:500,10,663–1-AP, Proteintech, Wuhan, China), cleaved-IL-1β (1:500, D3A3Z, Cell Signaling Technology, Massachusetts, USA), β-actin (1:2000, YM3028l, Immunoway, USA), β-tublin (1:2000, YM3030, Immunoway, USA), and GAPDH (1:2000; YM3029, Immunoway, USA).

For western blotting, cell or tissue extracts were made by homogenization in lysis buffer. About 20 μg of protein (determined by BCA assay) was boiled with Laemmli buffer. Whole-protein extracts were separated on 6–15% SDS–polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Merck Millipore, Darmstadt, Germany). After 1 h blocking procedure, the membranes were probed with primary antibodies in 1:1000 dilution. The antibodies for anti-NLRP3, ASC, caspase-4, caspase-5, pro-GSDMD, cleaved-GSDMD, LC3-II, flotillin-1, HSP70 and GAPDH antibodies were purchased from Cell Signaling (Beverly, MA, USA); anti-pro-caspase-1, cleaved-caspase-1 antibodies were purchased from Proteintech (Rosemont, IL 60018, USA); anti-Lamp-1, CD63 antibodies were purchased from Novus (Centennial CO 80112, USA); anti-TSG-101, p62 antibodies were purchased from Abcam Inc. (Cambridge, MA, USA). After the hybridization, membrane was washed with 1× TBST, and then probed with HRP-conjugated anti-mouse (Biolegend, San Diego, CA, USA) or anti-rabbit secondary antibodies (Jackson ImmunoResearch Lab Inc., West Grove, PA, USA) in 1:10,000 dilution. The immunoreactive proteins were visualized with Immobilon Western chemiluminescence HRP substrate (Merck, USA) and BioMax LightFilm (Eastman Kodak Company, New Heaven, CT, USA) according to the manufacturer’s instructions.

All human pulp samples from the healthy and pulpitis groups were demineralized in 10% EDTA for 3 months, dehydrated and rooted in paraffin to obtain 6 μm paraffin tissue sections. The tissue sections were incubated with primary antibody against cleaved GSDMD (#36425; Cell Signaling Technology, USA) for immunohistochemistry staining overnight at 4 °C. Incubation with secondary antibody and the subsequent colorization were performed as described in our previous research [20 (link)].

Samples and markers were applied to gels, which were run at 120 V. Subsequently, gels were blotted using the Trans-Blot Turbo Transfer System (BioRad, Berkely, CA, USA). Membranes were blocked for 1 h and primary antibodies ASC (B-3) (Santa Cruz Biotechnology, INC., Dallas, TX, USA; 1:500 in 5% dried milk), NLRP3 (Cell Signaling Technology, Danvers, MA, USA; 1:1000 in 5% BSA in TBST buffer), P-NFkB (Cell Signaling Technology; 1:1000 in 5% dried milk), cleaved Interleukin-1β (Cell Signaling Technology; 1:1000 in 5% dried milk), IL-1β (Cell Signaling Technology; 1:1000 in 5% BSA in TBST buffer), cleaved GSDMD (Cell Signaling Technology; 1:500 in 5% dried milk) or GSDMD (Merck; 1:500 in 5% dried milk) were added and incubated at 4 °C overnight. After washing, anti-mouse IgG or anti-rabbit IgG (both Cell Signaling Technology; 1:5000 in 5% dried milk) were added for 1 h. Membranes were developed using the Super Signal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and visualized using the FUSION SOLO S imaging system (Vilber Lourmat, Eberhardzell, Germany).