anti p mlkl  (Boster Bio)

Journal: Brain and Behavior

Article Title: Study on the Function and Mechanism of Neutrophil Extracellular Traps in Regulating Necroptosis Following Traumatic Brain Injury

doi: 10.1002/brb3.71275

Figure Lengend Snippet: Effects of Cl‐amidine and DNase I on necroptosis‐related protein expression after TBI. (A) Western blot analysis of RIP1, RIP3, MLKL, P‐RIP1, P‐RIP3, and P‐MLKL protein levels in the cortex of mice from Sham, TBI+Vehicle, TBI+Cl‐amidine, and TBI+DNase I groups. GAPDH was used as the loading control ( n = 4 per group). (B–G) Quantification of relative protein levels of RIP1, RIP3, MLKL, P‐RIP1, P‐RIP3, and P‐MLKL, normalized to GAPDH. Data are shown as mean ± SD ( n = 4 per group). Statistical significance is indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the indicated groups; ns: not significant.

Article Snippet: The membranes were blocked with 5% skimmed milk prepared in TBST (Tris‐buffered saline with 0.1% Tween‐20) at room temperature for 1 h. They were then incubated overnight at 4°C with the following primary antibodies: PAD4 (1:1000, 214810, Abcam), MPO (1:1000, ab208670, Abcam), Bcl‐2 (1:1000, A0208, Abclonal), Bax (1:1000, A19684, Abclonal), RIP1 (1:1000, #3493, Cell Signaling Technology), RIP3 (1:1000, #95702, Cell Signaling Technology), MLKL (1:1000, #37705, Cell Signaling Technology), P‐RIP1 (1:1000, #31122, Cell Signaling Technology), P‐RIP3 (1:1000, #91702, Cell Signaling Technology), P‐MLKL (1:1000, #37333, Cell Signaling Technology), and GAPDH (1:1000, AB‐P‐R001, GOODHERE Biotech).

Techniques: Expressing, Western Blot, Control

Journal: Journal of Advanced Research

Article Title: Cadmium targeting MLKL-Drp1 axis to trigger mitochondrial oxidative stress contributes to necroinflammation in rat kidney

doi: 10.1016/j.jare.2025.05.055

Figure Lengend Snippet: Cd triggered necroptosis in acute kidney injury. ( A ) Schematic protocol of Cd-induced acute kidney damage in rats. ( B-C ) Serum BUN and Cre levels in rat (mean ± SD, n = 6). ( D ) KIM-1 and NGAL protein levels in rat kidney tissues. ( E ) Kidney sections with H&E staining. ( F ) KEGG pathways enrichment data showed the related pathways in RNA-Seq dataset. ( G-H ) Differential genes associated with inflammation response and necroptosis. ( I ) NLRP3, Caspase-1, and IL-1β protein levels in rat kidney tissues. ( J ) RIPK1, RIPK3, p-MLKL, and MLKL protein levels in rat kidney tissues. ( K ) p-MLKL levels of immunofluorescence in rat kidney tissues. ( L ) Cleaved caspase 9, Cleaved caspase 8, and Cleaved caspase 3 protein levels in rat kidney tissues. * P < 0.05 and ** P < 0.01.

Article Snippet: Antibodies used were as follow: Caspase-8 (A0215), RIPK1 (A7414), RIPK3 (A5431), p-MLKL (AP0949), MLKL (A13451), HMGB1 (A2553), Caspase-1 (A0964), and IL-1β (A16288) were from ABclonal Technology Co.,Ltd.

Techniques: Staining, RNA Sequencing, Immunofluorescence

Journal: Journal of Advanced Research

Article Title: Cadmium targeting MLKL-Drp1 axis to trigger mitochondrial oxidative stress contributes to necroinflammation in rat kidney

doi: 10.1016/j.jare.2025.05.055

Figure Lengend Snippet: RIPK3/MLKL-mediated necroptosis was involved in Cd-induced cytotoxicity in NRK-52E cells. ( A ) Cells were exposure to 0, 5, 10, 20, 40 and 80 μM Cd for 12 or 24 h to detect cell viability by CCK-8 assay. ( B ) LDH release assay was performed after 12 or 24 h Cd treatment. ( C ) The morphology of cells exposed to different doses of Cd for 24 h was observed by a light microscope. ( D ) Representative flow cytometry images of cells dyed with PI/Annexin V-FITC. ( E ) Quantitative analysis of PI positive cells rate. ( F ) Ultrastructural morphology of cells exposed to 10 or 20 μM Cd for 24 h. ( G-I ) Cells were exposed to different doses of CdCl 2 for 24 h to analyze the protein levels of (G) RIPK1 and RIPK3, (H) p-MLK and MLKL, and (I) Cleaved caspase 3, Cleaved caspase 8, and Cleaved caspase 9. Cells were transfected with NC-siRNA or MLKL-siRNA. ( J ) Immunoblotting and ( K ) qRT-RCR were used to detect the knockdown efficiency at protein and mRNA levels, respectively. ( L-M ) CCK-8 assay and LDH release were applied to determine the effect of si-MLKL alone or combined with Cd treatment on the cytotoxicity of NRK-52E cells. * P < 0.05, ** P < 0.01.

Article Snippet: Antibodies used were as follow: Caspase-8 (A0215), RIPK1 (A7414), RIPK3 (A5431), p-MLKL (AP0949), MLKL (A13451), HMGB1 (A2553), Caspase-1 (A0964), and IL-1β (A16288) were from ABclonal Technology Co.,Ltd.

Techniques: CCK-8 Assay, Lactate Dehydrogenase Assay, Light Microscopy, Flow Cytometry, Transfection, Western Blot, Knockdown

Journal: Journal of Advanced Research

Article Title: Cadmium targeting MLKL-Drp1 axis to trigger mitochondrial oxidative stress contributes to necroinflammation in rat kidney

doi: 10.1016/j.jare.2025.05.055

Figure Lengend Snippet: Activated MLKL was responsible for Cd-triggered NLRP3 inflammasome cascade in NRK-52E cells. ( A , B ) Cells were exposed to different doses of CdCl 2 for 24 h to analyze the protein expressions of (A) NLRP3, pro-caspase-1, caspase-1, pro-IL-1β, IL-1β and (B) HMGB1. ( C ) Cells were exposed to different doses of CdCl 2 for 24 h to detect the mRNA expression levels of TNF-α , IL-1β , IL-2 , IL-6 and IL-18 using qRT-RCR assay. ( D ) NRK-52E cells transfected with NC-siRNA or MLKL-siRNA were exposed to 10 μM CdCl 2 for 24 h. qRT-RCR assay was conducted to detect the mRNA expression levels of TNF-α , IL-1β , IL-2 , IL-6 and IL-18. ( E , F ) NRK-52E cells transfected with NC-siRNA or MLKL-siRNA were exposed to 10 μM CdCl 2 for 24 h. Western blot was conducted to detect the protein expressions of (E) NLRP3, pro-caspase-1, caspase-1, pro-IL-1β, IL-1β and (F) HMGB1. * P < 0.05, ** P < 0.01.

Article Snippet: Antibodies used were as follow: Caspase-8 (A0215), RIPK1 (A7414), RIPK3 (A5431), p-MLKL (AP0949), MLKL (A13451), HMGB1 (A2553), Caspase-1 (A0964), and IL-1β (A16288) were from ABclonal Technology Co.,Ltd.

Techniques: Expressing, Transfection, Western Blot

Journal: Journal of Advanced Research

Article Title: Cadmium targeting MLKL-Drp1 axis to trigger mitochondrial oxidative stress contributes to necroinflammation in rat kidney

doi: 10.1016/j.jare.2025.05.055

Figure Lengend Snippet: Mitochondrial oxidative stress contributed to Cd-induced necroinflammation in NRK 52E cells. ( A ) Cells were labeled to IF staining with Tom20 (red) and p-MLKL (green) after treated with Cd for 12 and 24 h. ( B ) NRK-52E cells transfected with NC-siRNA or MLKL-siRNA were treated with 10 μM CdCl 2 for 24 h. Representative fluorescence images of JC-1 staining showed the transformation of JC-1 monomers (green) and aggregates (red). ( C ) Cellular ATP content was assessed in cells treated as above. ( D ) Representative fluorescence micrographs of MitoSOX Red staining, showing mtROS generation in cells treated as above. ( E-F ) CCK-8 assay and LDH release were applied to determine the effect of MT alone or combined with Cd treatment on the cytotoxicity of NRK-52E cells. ( G ) The mRNA levels of TNF-α, IL-1β, IL-2, IL-6 and IL-18 in MT-treated NRK-52E cells. ( H ) The protein expressions of NLRP3, pro-caspase-1, Caspase-1, pro-IL-1β, and IL-1β were detected by western blots in MT-treated NRK-52E cells. * P < 0.05, ** P < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Antibodies used were as follow: Caspase-8 (A0215), RIPK1 (A7414), RIPK3 (A5431), p-MLKL (AP0949), MLKL (A13451), HMGB1 (A2553), Caspase-1 (A0964), and IL-1β (A16288) were from ABclonal Technology Co.,Ltd.

Techniques: Labeling, Staining, Transfection, Fluorescence, Transformation Assay, CCK-8 Assay, Western Blot

Journal: Journal of Advanced Research

Article Title: Cadmium targeting MLKL-Drp1 axis to trigger mitochondrial oxidative stress contributes to necroinflammation in rat kidney

doi: 10.1016/j.jare.2025.05.055

Figure Lengend Snippet: Cd-activated MLKL interacted with Drp1 to promote mitochondrial oxidative stress in vitro. ( A ) Cross-enrichment of genes associated with the regulation of necroptotic process and the mitochondrial fission. ( B ) Calculation of FPKM values for Drp1 gene. ( C-D ) NRK-52E cells transfected with NC-siRNA or MLKL-siRNA were exposed to 10 μ M CdCl 2 for 24 h. Western blot was performed to assess the protein expressions of (C) Drp1 and Fis1, (D) p-Drp1(Ser616) and p-Drp1(Ser637). ( E ) Protein-protein docking of MLKL and Drp1 protein structure models. ( F ) Cells were labeled to IF staining with p-MLKL (red) and Drp1 (green) after treated with Cd for 12 and 24 h. ( G ) The mitochondrial fraction was immunoprecipitated with anti-MLKL or anti-Drp1 antibody, and then both IP and whole cell lysates were immunoblotted. * P < 0.05 and ** P < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Antibodies used were as follow: Caspase-8 (A0215), RIPK1 (A7414), RIPK3 (A5431), p-MLKL (AP0949), MLKL (A13451), HMGB1 (A2553), Caspase-1 (A0964), and IL-1β (A16288) were from ABclonal Technology Co.,Ltd.

Techniques: In Vitro, Transfection, Western Blot, Labeling, Staining, Immunoprecipitation

Journal: Journal of Advanced Research

Article Title: Cadmium targeting MLKL-Drp1 axis to trigger mitochondrial oxidative stress contributes to necroinflammation in rat kidney

doi: 10.1016/j.jare.2025.05.055

Figure Lengend Snippet: MLKL-decreased antioxidant capacity aggravated Cd-induced mitochondrial oxidative stress. NRK-52E cells transfected with NC-siRNA or MLKL-siRNA were treated with 10 μM CdCl 2 for 24 h. ( A-C ) The GSH/GSSG (A) and NADPH/NADP + ratios (B) were determined, and GSH-Px activity (C) was assessed. ( D-E ) Ca 2+ fluorescence probes were applied to detect the concentration of mitochondrial Ca 2+ (Rhod-2, AM) and cytoplasmic Ca 2+ (Fluo-4, FluO-4). ( F ) mPTP was detected in NRK-52E cells treated as above. * P < 0.05, ** P < 0.01.

Article Snippet: Antibodies used were as follow: Caspase-8 (A0215), RIPK1 (A7414), RIPK3 (A5431), p-MLKL (AP0949), MLKL (A13451), HMGB1 (A2553), Caspase-1 (A0964), and IL-1β (A16288) were from ABclonal Technology Co.,Ltd.

Techniques: Transfection, Activity Assay, Fluorescence, Concentration Assay