cleaved casp3 mouse mab  (Bioss)

Journal: MedComm

Article Title: Emestrin‐Type Epidithiodiketopiperazines Inhibited Gasdermin D‐Mediated Pyroptosis via Caspase‐3/7 Activation

doi: 10.1002/mco2.70548

Figure Lengend Snippet: Emestrin‐type ETPs inhibited pyroptosis in THP‐1 differentiated macrophages through activation of procaspase‐3/7. (A) Immunoblotting and Coomassie blue staining of cell lysates from THP‐1 differentiated macrophages treated with or without compound 2 (1 µM) 4 h post‐LPS and nigericin induction. P10 band (red rectangle) was sent for mass spectrum analysis. (B) N‑terminal sequence analysis result of p10 band. (C) Mechanism hypothesis of compounds 1 – 11 inhibiting pyroptosis. (D and E) Western blotting analyses of GSDMD, caspase‐3, caspase‐7 cleavage, and α‐tubulin in THP‐1 macrophages treated compound 2 (1 µM) prior and after 4 h pyroptosis induction with LPS and nigericin (mean ± SD; n = 3 per group, t ‐test, ** p < 0.01, **** p < 0.0001). (F) Western blotting analyses of caspase‐1, caspase‐3, caspase‐7 cleavage, and α‐tubulin in the THP‐1 differentiated macrophages treated with each compound (1 µM) 4 h after pyroptosis induction with LPS and nigericin. (G and H) Western blotting analyses of recombinant human procaspase‐3 protein 12 h postincubation with PAC‐1 and each compound (5 µM) respectively at 37 ℃. (I) The IC 50 result of these eleven compounds ( n = 3 per group). (J) In vitro activation of recombinant human procaspase‐3 protein by eleven compounds respectively ( n = 3 per group) and corresponding EC 50 values.

Article Snippet: Recombinant human procaspase‐3 protein (CUSABIO; Cat# CSB‐EP004548HU) was diluted to 200 ng/μL in activation buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM EDTA, 10% glycerol, and 0.1% CHAPS).

Techniques: Activation Assay, Western Blot, Staining, Sequencing, Recombinant, In Vitro

Journal: MedComm

Article Title: Emestrin‐Type Epidithiodiketopiperazines Inhibited Gasdermin D‐Mediated Pyroptosis via Caspase‐3/7 Activation

doi: 10.1002/mco2.70548

Figure Lengend Snippet: Emestrin‐type ETPs directly bound to human procaspase‐3 protein. (A) Molecular docking analyses of compounds 1 and 3 – 11 with human procaspase‐3 protein (PDB ID: 3DEK). (B) Molecular docking analysis of compound 2 with human procaspase‐3 protein. (C) Compound binding activity to recombinant human procaspase‐3 protein measured by SPR. (D and E) SPR test of compound 2 interacting with recombinant human procaspase‐3 protein. The K d value is 76.72 µM. (F and G) Western blotting analyses of recombinant human procaspase‐3 protein different time points after incubation with PAC‐1 (5 µM) and compound 2 (5 µM) (mean ± SD; n = 3 per group, t ‐test, ** p < 0.01). (H and I) CETSA results for compound 2 (5 µM) in THP‐1 differentiated macrophage lysates.

Article Snippet: Recombinant human procaspase‐3 protein (CUSABIO; Cat# CSB‐EP004548HU) was diluted to 200 ng/μL in activation buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM EDTA, 10% glycerol, and 0.1% CHAPS).

Techniques: Binding Assay, Activity Assay, Recombinant, Western Blot, Incubation

Journal: Poultry Science

Article Title: Effect of selenium-enriched Ganoderma lucidum powder in testicular tissues, oxidative stress biomarkers and antiapoptotic capacities in roosters

doi: 10.1016/j.psj.2025.106185

Figure Lengend Snippet: SeGL attenuates the level of apoptosis in the rooster testes. A–B . Relative mRNA expression of Bax, Bcl-2, Caspase3 and the Bax/Bcl-2 ratio in the testes of the control, LSe, MSe, or HSe group (n = 3). C . Western blot of Bax, Bcl-2, and Caspase3 proteins in the testes of each group. D–E . Quantitative analysis of Bax, Bcl-2, Caspase3 protein expression and the Bax/Bcl-2 ratio in the testes of each group (n = 3). Data are presented as means ± SD; different lowercase letters indicate significant differences between groups (P < 0.05).

Article Snippet: After blocking with 5 % non-fat milk (A600669, Sangon, Shanghai, China) in TBST (Tris-buffered saline with 0.1 % Tween-20) for 1 h at room temperature, membranes were incubated overnight at 4°C with primary antibodies against target proteins [Bax (50599-2-lg, Proteintech, Wuhan, China), Bcl-2 (26593-1-AP, Proteintech, Wuhan, China), Caspase3 (PB9188, Boster, Wuhan, China), NF-κB (PB9149, Boster, Wuhan, China), p53 (PB9008, Boster, Wuhan, China), StAR (A00051-1, Boster, Wuhan, China), Cyp11a1 (BA3699, Boster, Wuhan, China), Hsd3b1 (A02856-2, Boster, Wuhan, China)] and β-tubulin (internal control; bsm-33034 M, Bioss Antibodies, Beijing, China).

Techniques: Expressing, Control, Western Blot

Journal: Oncology Letters

Article Title: Transferrin receptor serves a role in promoting PANoptosis in thyroid cancer

doi: 10.3892/ol.2025.15394

Figure Lengend Snippet: TFRC contributes to PANoptosis in TC cells. Effects of (A) PGMLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro and (B) shRNA transfection on the expression of TFRC mRNA in BCPAP and K1 cells, assessed using reverse transcription-quantitative PCR assay. (C) PI/Calcein-AM staining was performed to assess the effect of TFRC on cell death in BCPAP (OV-NC and OV-TFRC groups) and K1 (KD-NC and KD-TFRC groups) cells. PI (red) and Calcein-AM (green) label dead and viable cells, respectively. (D) YO-PRO-1/PI staining was performed to evaluate pyroptosis, apoptosis and necroptosis in BCPAP and K1 cells following exogenous modulation of TFRC expression. YO-PRO-1 (green) indicates apoptotic or necroptotic cells, whereas PI (red) labels necroptotic or pyroptotic cells. (E) Western blot analysis of markers associated with pyroptosis (cleaved-CASP1/CASP1), apoptosis (cleaved-CASP3/CASP3) and necroptosis (pMLKL/tMLKL and tRIPK3), with corresponding quantitative analyses. TFRC, transferrin receptor; sh, short hairpin; OV, overexpression; NC, negative control; KD, knockdown; CASP; caspase; MLKL, mixed lineage kinase domain like pseudokinase; pMLKL, phospho-MLKL; tMLKL, total-MLKL; tRIPK3, total receptor interacting serine/threonine kinase 3.

Article Snippet: Membranes were blocked with 5% skimmed milk at room temperature for 1 h and incubated with the following primary antibodies overnight at 4°C: Vinculin mouse mAb (1:50,000; cat. no. 66305-1-Ig; Proteintech Group, Inc.), CASP3 mouse mAb (1:2,000; cat. no. bsm-33284M; BIOSS), cleaved-CASP3 mouse mAb (1:2,000; cat. no. bsm-33199M; BIOSS), CASP1 rabbit pAb (1:2,000; cat. no. 22915-1-AP; Proteintech Group, Inc.), cleaved-CASP1 rabbit pAb (1:3,000; cat. no. PA5-77886; Thermo Fisher Scientific, Inc.), phospho-(p)MLKL rabbit pAb (1:2,000; cat. no. PA5-105677; Thermo Fisher Scientific, Inc.), total-(t)MLKL mouse mAb (1:50,000; cat. no. 66675-1-Ig; Proteintech Group, Inc.) and tRIPK3 rabbit pAb (1:10,000; cat. no. 29080-1-AP; Proteintech Group, Inc.).

Techniques: shRNA, Transfection, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Staining, Western Blot, Over Expression, Negative Control, Knockdown

Journal: Oncology Letters

Article Title: Transferrin receptor serves a role in promoting PANoptosis in thyroid cancer

doi: 10.3892/ol.2025.15394

Figure Lengend Snippet: TFRC contributes to PANoptosis in TC cells. Effects of (A) PGMLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro and (B) shRNA transfection on the expression of TFRC mRNA in BCPAP and K1 cells, assessed using reverse transcription-quantitative PCR assay. (C) PI/Calcein-AM staining was performed to assess the effect of TFRC on cell death in BCPAP (OV-NC and OV-TFRC groups) and K1 (KD-NC and KD-TFRC groups) cells. PI (red) and Calcein-AM (green) label dead and viable cells, respectively. (D) YO-PRO-1/PI staining was performed to evaluate pyroptosis, apoptosis and necroptosis in BCPAP and K1 cells following exogenous modulation of TFRC expression. YO-PRO-1 (green) indicates apoptotic or necroptotic cells, whereas PI (red) labels necroptotic or pyroptotic cells. (E) Western blot analysis of markers associated with pyroptosis (cleaved-CASP1/CASP1), apoptosis (cleaved-CASP3/CASP3) and necroptosis (pMLKL/tMLKL and tRIPK3), with corresponding quantitative analyses. TFRC, transferrin receptor; sh, short hairpin; OV, overexpression; NC, negative control; KD, knockdown; CASP; caspase; MLKL, mixed lineage kinase domain like pseudokinase; pMLKL, phospho-MLKL; tMLKL, total-MLKL; tRIPK3, total receptor interacting serine/threonine kinase 3.

Article Snippet: Membranes were blocked with 5% skimmed milk at room temperature for 1 h and incubated with the following primary antibodies overnight at 4°C: Vinculin mouse mAb (1:50,000; cat. no. 66305-1-Ig; Proteintech Group, Inc.), CASP3 mouse mAb (1:2,000; cat. no. bsm-33284M; BIOSS), cleaved-CASP3 mouse mAb (1:2,000; cat. no. bsm-33199M; BIOSS), CASP1 rabbit pAb (1:2,000; cat. no. 22915-1-AP; Proteintech Group, Inc.), cleaved-CASP1 rabbit pAb (1:3,000; cat. no. PA5-77886; Thermo Fisher Scientific, Inc.), phospho-(p)MLKL rabbit pAb (1:2,000; cat. no. PA5-105677; Thermo Fisher Scientific, Inc.), total-(t)MLKL mouse mAb (1:50,000; cat. no. 66675-1-Ig; Proteintech Group, Inc.) and tRIPK3 rabbit pAb (1:10,000; cat. no. 29080-1-AP; Proteintech Group, Inc.).

Techniques: shRNA, Transfection, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Staining, Western Blot, Over Expression, Negative Control, Knockdown