primary antibodies against gsdmd  (ABclonal Biotechnology)

Journal: Poultry Science

Article Title: Selenoprotein S ablation-mediated pyroptosis contributes to liver damage resulting from selenium deficiency in chickens

doi: 10.1016/j.psj.2025.105269

Figure Lengend Snippet: SELS knockdown induces pyroptosis. (A-B) Western blot and qRT-PCR shows protein and transcription levels of SELS in primary hepatocytes and LMH cells. (C) Immunofluorescence images and quantification of NLRP3 and GSDMD in primary hepatocytes. (D-E) Western blot shows protein levels of pyroptosis-related genes in primary hepatocytes and LMH cells. (F) QRT-PCR detects transcript levels of pyroptosis-related genes in primary hepatocytes and LMH cells. All data were biologically replicated ( n = 3). A significant difference " p < 0.05″ is indicated by "*", otherwise " p > 0.05″ is labeled "ns", when compared with control group using t-test. Bars labeled with different letters indicate significant differences screened by one-way ANOVA.

Article Snippet: After blocking with 5 % BSA for 30 min, primary antibodies against NLRP3 (ABclonal, A12694, 1:100) or GSDMD (ABclonal, A24476 , 1:200) were added overnight at 4 °C followed by incubation with HRP-conjugated secondary antibody (Servicebio, GB23303, 1:200) for 45 min. After treatment with 3,3′-diaminobenzidine solution (Servicebio, G1212), images were captured using a fluorescence microscope.

Techniques: Knockdown, Western Blot, Quantitative RT-PCR, Immunofluorescence, Labeling, Control

Journal: Poultry Science

Article Title: Selenoprotein S ablation-mediated pyroptosis contributes to liver damage resulting from selenium deficiency in chickens

doi: 10.1016/j.psj.2025.105269

Figure Lengend Snippet: NF-κB is the upstream pathway of pyroptosis. (A-B) Western blot and qRT-PCR shows protein and transcription levels of pyroptosis-related genes in primary hepatocytes. (C-D) Western blot and qRT-PCR displays protein and transcription levels of pyroptosis-related genes in LMH cells. (E-F) Immunofluorescence images and quantification of NLRP3 and GSDMD in primary hepatocytes. All data were biologically replicated ( n = 3). Bars labeled with different letters indicate significant differences screened by one-way ANOVA.

Article Snippet: After blocking with 5 % BSA for 30 min, primary antibodies against NLRP3 (ABclonal, A12694, 1:100) or GSDMD (ABclonal, A24476 , 1:200) were added overnight at 4 °C followed by incubation with HRP-conjugated secondary antibody (Servicebio, GB23303, 1:200) for 45 min. After treatment with 3,3′-diaminobenzidine solution (Servicebio, G1212), images were captured using a fluorescence microscope.

Techniques: Western Blot, Quantitative RT-PCR, Immunofluorescence, Labeling

Journal: Poultry Science

Article Title: Selenoprotein S ablation-mediated pyroptosis contributes to liver damage resulting from selenium deficiency in chickens

doi: 10.1016/j.psj.2025.105269

Figure Lengend Snippet: ROS regulates pyroptosis of hepatocytes. (A-B) Immunofluorescence images and quantification of NLRP3 and GSDMD in hepatocytes. (C-D) Western blot shows protein levels of pyroptosis-related genes in hepatocytes and LMH cells. (E-F) QRT-PCR detects transcription levels of pyroptosis-related genes in hepatocytes and LMH cells. (G-H) The mRNA expression of the NF-κB pathway in primary hepatocytes and LMH cells. All data were biologically replicated ( n = 3). Bars labeled with different letters indicate significant differences screened by one-way ANOVA.

Article Snippet: After blocking with 5 % BSA for 30 min, primary antibodies against NLRP3 (ABclonal, A12694, 1:100) or GSDMD (ABclonal, A24476 , 1:200) were added overnight at 4 °C followed by incubation with HRP-conjugated secondary antibody (Servicebio, GB23303, 1:200) for 45 min. After treatment with 3,3′-diaminobenzidine solution (Servicebio, G1212), images were captured using a fluorescence microscope.

Techniques: Immunofluorescence, Western Blot, Quantitative RT-PCR, Expressing, Labeling

Journal: European Journal of Medical Research

Article Title: High-fat diet activates pyroptosis of retinal pigment epithelial cells in aged TgAPPswePS1 transgenic mice

doi: 10.1186/s40001-025-02898-5

Figure Lengend Snippet: Immunofluorescence analysis of the pyroptosis-related proteins in RPE cells. Scale bar = 20 μm. A Representative images showing the expressions of NLRP3, caspase-1 and GSDMD in RPE cells. B Representative images showing the expressions of IL-1β and IL-18 in RPE cells. C Semi-quantitative analysis of fluorescence intensity for NLRP3, caspase-1, GSDMD, IL-1β and IL-18 expressions in RPE cells. ** p < 0.01, ***p < 0.001, ****p < 0.0001, ns, no significance

Article Snippet: After blocking with 5% nonfat dry milk, membranes were incubated overnight at 4 °C with primary antibodies against NLRP3 (1:1000, #15101), Pro-Caspase-1 (1:1000, #24232), Cleaved-Caspase-1 (1:1000, #89332), Cleaved-GSDMD (1:1000, #10137, Cell Signaling Technology, MA, USA), GSDMD (1:1000, ab219800) and β-actin (1:1000, ab8226, Abcam Biotechnology, UK).

Techniques: Immunofluorescence, Fluorescence

Journal: European Journal of Medical Research

Article Title: High-fat diet activates pyroptosis of retinal pigment epithelial cells in aged TgAPPswePS1 transgenic mice

doi: 10.1186/s40001-025-02898-5

Figure Lengend Snippet: Expression alternations of pyroptosis-related proteins in RPE cells. A NLRP3, caspase-1, GSDMD protein expressions in RPE were detected via western blotting. B IL-1β and IL-18 levels in RPE were tested via ELISA. * p < 0.05, **p < 0.01, ***p < 0.001, ns, no significance

Article Snippet: After blocking with 5% nonfat dry milk, membranes were incubated overnight at 4 °C with primary antibodies against NLRP3 (1:1000, #15101), Pro-Caspase-1 (1:1000, #24232), Cleaved-Caspase-1 (1:1000, #89332), Cleaved-GSDMD (1:1000, #10137, Cell Signaling Technology, MA, USA), GSDMD (1:1000, ab219800) and β-actin (1:1000, ab8226, Abcam Biotechnology, UK).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Acta Neuropathologica Communications

Article Title: Transient receptor potential vanilloid 4 blockage attenuates pyroptosis in hippocampus of mice following pilocarpine‑induced status epilepticus

doi: 10.1186/s40478-025-01990-5

Figure Lengend Snippet: Changes in calpain 1–NLRP3/cas-1–GSDMD pathway in the hippocampus of PISE mice. (A) The inactive/total calpain 1 ratio markedly decreased (independent-samples t test, t = 9.42, P < 0.01) while the inactive/total calpain 2 ratio did not change (independent-samples t test, t = − 0.16, P = 0.87) in the hippocampus of PISE mice. (B) The protein levels of NLRP3 (Mann–Whitney U test, P < 0.01), c-cas-1 (Mann–Whitney U test, P < 0.01), IL-1β (Mann–Whitney U test, P < 0.01), and N-GSDMD (Mann–Whitney U test, P < 0.01) increased markedly in the hippocampus of PISE mice. (C) The number of GSDMD + cells (red arrow) increased markedly in the hippocampus in PISE mice (independent-samples t test, t = − 18.01, P < 0.01). (D) GSDMD + /NeuN + (D-i, white arrow), GSDMD + /GFAP + (D-ii, white star) and GSDMD + /Iba-1 + (D-iii, white triangle) cells in the hippocampus of control and PISE mice. Scale = 50 μm. ×4 objective for the top row in D-i, D-ii and D-iii; ×10 objective for C-DAPI, C-GSDMD, C-Merge; ×20 objective for C-enlarge and the down row in D-i, D-ii and D-iii. ** P < 0.01 vs. control

Article Snippet: For double immunostaining, free-floating sections were incubated with primary antibodies against GSDMD (cat. no. AF-4012, 1:100, Affinity Biosciences, Melbourne, Australia), neuronal nuclei (NeuN, cat. no. 26975-1-AP, 1:500, Proteintech Group Inc, Wuhan, China), glial fibrillar acidic protein (GFAP, cat. no. MAB360, 1:500, Millipore, Massachusetts, USA), and ionized calcium-binding adapter molecule 1 (Iba-1, cat. no. Ab178847, 1:500, Abcam, Cambridge, UK).

Techniques: MANN-WHITNEY, Control

Journal: Acta Neuropathologica Communications

Article Title: Transient receptor potential vanilloid 4 blockage attenuates pyroptosis in hippocampus of mice following pilocarpine‑induced status epilepticus

doi: 10.1186/s40478-025-01990-5

Figure Lengend Snippet: Effect of calpain inhibitor MDL-28170 on calpain 1–NLRP3/cas-1–GSDMD pathway in the hippocampus of PISE mice. (A) The calpain inhibitor MDL-28170 markedly increased the inactive/total calpain 1 rato in the hippocampus of PISE mice (Mann–Whitney U test, P < 0.01). (B) MDL-28170 did not affect NLRP3 protein levels in the hippocampus of PISE mice (independent-samples t test, t = 0.74, P = 0.47), but decreased c-cas-1 (independent-samples t test, t = 7.81, P < 0.01), IL-1β (independent-samples t test, t = 6.30, P < 0.01), and N-GSDMD protein levels (Mann–Whitney U test, P < 0.01). (C) MDL-28170 increased GSDMD + cells (red arrow) number in the hippocampus of PISE mice (independent-samples t test, t = 8.83, P < 0.01). (D) GSDMD + /NeuN + (D-i, white arrow), GSDMD + /GFAP + (D-ii, white star) and GSDMD + /Iba-1 + (D-iii, white triangle) cells in the hippocampus of vehicle- and MDL-28179-treated PISE mice. (E) The numbers of surviving pyramidal neurons reduced in the hippocampal CA1 (independent-samples t test, t = 22.76, P < 0.01) and CA2/3 (independent-samples t test, t = 28.02, P < 0.01) area of PISE mice (E-i). Administration of MDL-28170 to PISE mice increased the numbers of surviving pyramidal cells in the hippocampal CA1 (independent-samples t test, t = 6.88, P < 0.01) and CA2/3 area (independent-samples t test, t = 6.15, P < 0.01) (E-ii). Scale = 50 μm. ×4 objective for the top row in D-i, D-ii and D-iii; ×10 objective for C-DAPI, C-GSDMD, C-Merge, and the middle column in E-i and E-ii; ×20 objective for C-enlarge, the down row in D-i, D-ii, and D-iii; ×40 objective for the left and right columns in E-i and E-ii. ## P < 0.01 vs. PISE + vehicle (ip.), ** P < 0.01 vs. control

Article Snippet: For double immunostaining, free-floating sections were incubated with primary antibodies against GSDMD (cat. no. AF-4012, 1:100, Affinity Biosciences, Melbourne, Australia), neuronal nuclei (NeuN, cat. no. 26975-1-AP, 1:500, Proteintech Group Inc, Wuhan, China), glial fibrillar acidic protein (GFAP, cat. no. MAB360, 1:500, Millipore, Massachusetts, USA), and ionized calcium-binding adapter molecule 1 (Iba-1, cat. no. Ab178847, 1:500, Abcam, Cambridge, UK).

Techniques: MANN-WHITNEY, Control

Journal: Acta Neuropathologica Communications

Article Title: Transient receptor potential vanilloid 4 blockage attenuates pyroptosis in hippocampus of mice following pilocarpine‑induced status epilepticus

doi: 10.1186/s40478-025-01990-5

Figure Lengend Snippet: Effect of TRPV4 antagonist on calpain 1–NLRP3/cas-1–GSDMD pathway in the hippocampus of PISE mice. A and C . Administration of TRPV4 antagonist HC-067047 increased inactive/total calpain 1 ratio in the hippocampus of control (A, Mann–Whitney U test, P < 0.01) and PISE mice (C, independent-samples t test, t = − 17.09, P < 0.01). B and D . Administration of HC-067047 decreased NLRP3 (B, HC-067047: independent-samples t test, t = 6.49, P < 0.01; D, PISE + HC-067047: Mann–Whitney U test, P < 0.01), c-cas-1 (B, HC-067047: independent-samples t test, t = 6.53, P < 0.01; D, PISE + HC-067047: independent-samples t test, t = 9.38, P < 0.01), IL-1β (B, HC-067047: independent-samples t test, t = 4.23, P < 0.01; D, PISE + HC-067047: independent-samples t test, t = 12.31, P < 0.01), and N-GSDMD protein levels (B, HC-067047: independent-samples t test, t = 5.28, P < 0.01; D, PISE + HC-067047: Mann–Whitney U test, P < 0.01). E and F . Administration of HC-067047 decreased GSDMD + cells (red arrow) numbers in the hippocampus of control (E-i, HC-067047: independent-samples t test, t = 4.34, P < 0.01) and PISE mice (F-i, PISE + HC-067047: Mann–Whitney U test, P < 0.01; white star). GSDMD + /NeuN + (white arrow), GSDMD + /GFAP + (white star) and GSDMD + /Iba-1 + (white triangle) cells in the hippocampus in control (E-ii, E-iii and E-iv) and PISE mice (F-ii, F-iii and F-iv) treated with either vehicle or HC-067047. G. HC-067047 treatment increased the numbers of surviving neurons in the hippocampal CA1 (G-i, independent-samples t test, t = 5.14, P < 0.01; G-ii, independent-samples t test, t = 10.12, P < 0.01) and CA2/3 (G-i, independent-samples t test, t = 2.35, P = 0.03; G-ii, independent-samples t test, t = 14.20, P < 0.01) area of control (G-i) and PISE mice (G-ii). Scale = 50 μm. ×4 objective for the top row in E-ii, E-iii, E-vi, F-ii, F-iii, F-vi; ×10 objective for E-i-DAPI, E-i-GSDMD, E-i-Merge, F-i-DAPI, F-i-GSDMD, F-i-Merge, and the middle columns in G-i and G-ii; ×20 objective for E-i-enlarge, F-i-enlarge, the down row in E-ii, E-iii, E-vi, F-ii, F-iii and F-vi; ×40 objective for the left and right columns in G-i, and G-ii. < P < 0.05, << P < 0.01 vs. control (icv.), && P < 0.01 vs. PISE + vehicle (icv.)

Article Snippet: For double immunostaining, free-floating sections were incubated with primary antibodies against GSDMD (cat. no. AF-4012, 1:100, Affinity Biosciences, Melbourne, Australia), neuronal nuclei (NeuN, cat. no. 26975-1-AP, 1:500, Proteintech Group Inc, Wuhan, China), glial fibrillar acidic protein (GFAP, cat. no. MAB360, 1:500, Millipore, Massachusetts, USA), and ionized calcium-binding adapter molecule 1 (Iba-1, cat. no. Ab178847, 1:500, Abcam, Cambridge, UK).

Techniques: Control, MANN-WHITNEY

Journal: Acta Neuropathologica Communications

Article Title: Transient receptor potential vanilloid 4 blockage attenuates pyroptosis in hippocampus of mice following pilocarpine‑induced status epilepticus

doi: 10.1186/s40478-025-01990-5

Figure Lengend Snippet: Effect of TRPV4 agonist on calpain 1-NLRP3/cas-1-GSDMD pathway in the hippocampus. A . The TRPV4 agonist GSK1016790A decreased inactive/total calpain 1 ratio (independent-samples t test, t = 12.72, P < 0.01) but did not change inactive/total calpain 2 ratio (independent-samples t test, t = 0.19, P = 0.86) in the hippocampus. B and C . GSK1016790A increased NLRP3 (B, Mann–Whitney U test, P < 0.01), c-cas-1 (B, Mann–Whitney U test, P < 0.01), IL-1β (B, Mann–Whitney U test, P < 0.01), and N-GSDMD (B, Mann–Whitney U test, P < 0.01) protein levels, and GSDMD + cells (red arrow) number (C, independent-samples t test, t = − 20.96, P < 0.01) in the hippocampus. D . GSDMD + /NeuN + (D-i, white arrow), GSDMD + /GFAP + (D-ii, white star) and GSDMD + /Iba-1 + (D-iii, white triangle) cells in the hippocampus of control and GSK1016790A-injected mice. E and F . The calpain inhibitor MDL-28170 increased inactive/total calpain 1 ratio (E, Mann–Whitney U test, P < 0.01) and decreased c-cas-1 (F, independent-samples t test, t = 8.73, P < 0.01), IL-1β (F, independent-samples t test, t = 9.35, P < 0.01), and N-GSDMD protein levels (F, independent-samples t test, t = 13.52, P < 0.01) but did not affect NLRP3 protein levels (F, independent-samples t test, t = − 0.20, P = 0.85) in the hippocampus of GSK1016790A-injected mice. G and H . The NLRP3 inhibitor MCC950 (G) and cas-1 inhibitor Ac-YVAD-cmk (H) decreased c-cas-1 (G, independent-samples t test, t = 8.17, P < 0.01; H, Mann–Whitney U test, P < 0.01), IL-1β (G, independent-samples t test, t = 11.62, P < 0.01; H, Mann–Whitney U test, P < 0.01) and N-GSDMD protein levels (G, Mann–Whitney U test, P < 0.01; H, independent-samples t test, t = 13.38, P < 0.01) in the hippocampus in GSK1016790A-injected mice. Scale = 50 μm. ×4 objective for the top row in D-i, D-ii and D-iii; ×10 objective for C-DAPI, C-GSDMD and C-Merge; ×20 objective for C-enlarge, the down row in D-i, D-ii and D-iii. << P < 0.01 vs. Control (icv.), $$ P < 0.01 vs. GSK1016790A + vehicle, %% P < 0.01 vs. GSK1016790A + vehicle

Article Snippet: For double immunostaining, free-floating sections were incubated with primary antibodies against GSDMD (cat. no. AF-4012, 1:100, Affinity Biosciences, Melbourne, Australia), neuronal nuclei (NeuN, cat. no. 26975-1-AP, 1:500, Proteintech Group Inc, Wuhan, China), glial fibrillar acidic protein (GFAP, cat. no. MAB360, 1:500, Millipore, Massachusetts, USA), and ionized calcium-binding adapter molecule 1 (Iba-1, cat. no. Ab178847, 1:500, Abcam, Cambridge, UK).

Techniques: MANN-WHITNEY, Control, Injection