anti human total gsdmd mouse monoclonal antibody mab  (Santa Cruz Biotechnology)

Journal: Journal of reproductive immunology

Article Title: Human fetal membrane IL-1β production in response to bacterial components is mediated by uric-acid induced NLRP3 inflammasome activation.

doi: 10.1016/j.jri.2021.103457

Figure Lengend Snippet: Fig. 3. FM secretion of IL-1β and IL-18 in response to bacterial components is mediated through a non-pyroptotic mechanism. Intact human FM explants from 3-8 patients were untreated or treated with either LPS; PGN; or MDP. (A) After 24 h, cell-free culture supernatants were collected and measured for LDH release (n = 8). (B) After 24 h, FM explants were homogenized for protein and expression of cleaved and total GSDMD evaluated by Western blot. Image shows one representative blot while the bar chart shows quantification of protein expression as determined by densitometry (n = 3).

Article Snippet: Membranes were probed with either the anti-human total GSDMD mouse monoclonal antibody (mAb) (64-Y) (1:1000, sc81868; Santa Cruz Biotechnology, Inc, Dallas, TX) or the anti-human cleaved GSDMD (Asp275) rabbit mAb (1:500, E7H9G; Cell Signaling Technology, Danvers, MA). β-Actin was used as a loading control (1:5000, Sigma-Aldrich).

Techniques: Expressing, Western Blot

Journal: Frontiers in endocrinology

Article Title: Excessive Iodine Promotes Pyroptosis of Thyroid Follicular Epithelial Cells in Hashimoto's Thyroiditis Through the ROS-NF-κB-NLRP3 Pathway.

doi: 10.3389/fendo.2019.00778

Figure Lengend Snippet: FIGURE 1 | Aberrant pyroptosis occurs in the thyroid tissues of HT patients and is induced by excessive iodine in vitro. (A,B) Gasdermin D (GSDMD) protein levels in the thyroid tissue of HT patients (n = 20) and controls (n = 10) were analyzed by immunoblots. “Control” indicates tissues from patients with nodular goiter of the thyroid. Representative immunoblotting results and quantification of GSDMD are shown. (C,D) Representative results of GSDMD immunohistochemical staining in HT tissues (n = 20) and control tissues (n = 10) are shown. Brown regions represent positive expression (original magnification, ×200; scale bars, 100 µm). (E–G) Nthy-ori3-1 cells were harvested after treatment with a gradient of concentrations of sodium iodide (NaI) for 24 h. The images presented are immunoblots probed for GSDMD (Hsp60 served as the loading control). All statistical results shown are representative of three replicates. (H) The cell viability of Nthy-ori 3-1 cells was assessed by CCK-8 assays after NaI treatment for 24 h. All statistical results shown are representative of three replicates. Significant differences and P-values were calculated by unpaired t-tests or one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Thyroid sections were blocked with 2% bovine serum albumin in PBS for 30min and then incubated with mouse anti-human GSDMD or mouse antihuman IL-1β antibodies (Santa Cruz, NJ, USA) at 4◦C overnight.

Techniques: In Vitro, Western Blot, Control, Immunohistochemical staining, Staining, Expressing, CCK-8 Assay

Journal: Frontiers in endocrinology

Article Title: Excessive Iodine Promotes Pyroptosis of Thyroid Follicular Epithelial Cells in Hashimoto's Thyroiditis Through the ROS-NF-κB-NLRP3 Pathway.

doi: 10.3389/fendo.2019.00778

Figure Lengend Snippet: FIGURE 3 | The NLRP3 inflammasome participates in excessive iodine-induced pyroptosis in TFCs. (A,B) The NLRP3 expression levels were measured by immunoblots when Nthy-ori 3-1 cells were treated with NaI with or without NAC (10 mM) or IKK-16 (2 µM) for 24 h. (C,D) Verification of the silencing efficiency of NLRP3 by siRNA in Nthy-ori3-1 cells is shown by immunoblots; NC indicates the negative control. (E,F) The protein levels of GSDMD were detected by immunoblots after transfection of siNLRP3 in NaI-treated Nthy-ori 3-1 cells. All statistical results shown are representative of three replicates. Significant differences and P-values were calculated by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Thyroid sections were blocked with 2% bovine serum albumin in PBS for 30min and then incubated with mouse anti-human GSDMD or mouse antihuman IL-1β antibodies (Santa Cruz, NJ, USA) at 4◦C overnight.

Techniques: Expressing, Western Blot, Negative Control, Transfection

Journal: Frontiers in endocrinology

Article Title: Excessive Iodine Promotes Pyroptosis of Thyroid Follicular Epithelial Cells in Hashimoto's Thyroiditis Through the ROS-NF-κB-NLRP3 Pathway.

doi: 10.3389/fendo.2019.00778

Figure Lengend Snippet: FIGURE 5 | Proposed model of TFC pyroptosis in excessive iodine-promoted HT. Excessive iodine enters TFCs and induces the production of ROS, which activates NF-κB signaling and increases GSDMD-FL. In addition, the NLRP3 inflammasome is activated by NF-κB signaling, leading to GSDMD-FL cleavage into GSDMD-N, which triggers pore formation in the membrane and the release of mature IL-1β from cells, causing a sterile inflammatory response and further contributing to pyroptotic cell death and the subsequent promotion of HT.

Article Snippet: Thyroid sections were blocked with 2% bovine serum albumin in PBS for 30min and then incubated with mouse anti-human GSDMD or mouse antihuman IL-1β antibodies (Santa Cruz, NJ, USA) at 4◦C overnight.

Techniques: Membrane, Sterility

Journal: Cell Reports

Article Title: Enteropathogenic Escherichia coli Stimulates Effector-Driven Rapid Caspase-4 Activation in Human Macrophages

doi: 10.1016/j.celrep.2019.03.100

Figure Lengend Snippet: EPEC-Induced Caspase-1 Activation, Cytokine Processing, and Pyroptosis Requires Caspase-4 and GSDMD (A and B) Real-time PI-uptake assays from THP1 cells stably expressing non-targeting ( CTRL ) or miRNA30E against the indicated genes. THP1 cells were infected with EPEC for indicated times (A) or transfected with LPS using lipofectamine 2000 (B). Means ± SEMs from n = 3 independent experiments are shown. ∗∗∗∗ p < 0.0001 by two-way ANOVA with FDR-based correction for multiple comparisons for indicated comparisons between CTRL miR and others. (C–E) Representative immunoblots from THP1 cells stably expressing non-targeting ( CTRL ) or miRNA30E against the indicated genes transfected with LPS for 4 h (C) or infected with EPEC for 4 h (D and E). Pooled supernatants and lysates were used for immunoblots. Schematics in (C) and (E) show caspase-4-dependent inflammasome signaling by transfected LPS and EPEC, respectively. Data in (C)–(E) are representative of experiments performed at least three times.

Article Snippet: Mouse Anti-Human GSDMDC1 Monoclonal Antibody, Unconjugated, Clone 64-Y , Santa Cruz Biotechnology , Cat# sc-81868, RRID: AB_2263768.

Techniques: Activation Assay, Stable Transfection, Expressing, Infection, Transfection, Western Blot

Journal: Cell Reports

Article Title: Enteropathogenic Escherichia coli Stimulates Effector-Driven Rapid Caspase-4 Activation in Human Macrophages

doi: 10.1016/j.celrep.2019.03.100

Figure Lengend Snippet:

Article Snippet: Mouse Anti-Human GSDMDC1 Monoclonal Antibody, Unconjugated, Clone 64-Y , Santa Cruz Biotechnology , Cat# sc-81868, RRID: AB_2263768.

Techniques: Produced, Recombinant, Transfection, Protease Inhibitor, Western Blot, Staining, Cytotoxicity Assay, Software