Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Wogonin protects against cisplatin-induced acute kidney injury by targeting RIPK1-mediated necroptosis.

doi: 10.1038/labinvest.2017.115

Figure Lengend Snippet: Figure 5 Wogonin fails to further reduce cisplatin-induced cell injury and inflammatory response in HK2 cells where RIPK1 is blocked. (a) Western blot analysis and quantitative data of RIPK1/RIPK3/MLKL signaling in Nec-1-treated HK2 cells. (b) Western blot analysis and quantitative data of KIM-1. Results indicated that when RIPK1 was blocked, wogonin failed to further suppress the protein levels of KIM-1. (c) Real-time PCR showed that when RIPK1 was blocked, wogonin failed to further decrease IL-6 in mRNA level. Independent experiments were performed throughout the in vitro studies in triplicate or quadruplicate. *Po0.05, **Po0.01, ***Po0.001 compared with the control. ##Po0.01, ###Po0.001 compared with cisplatin-treated group. $$Po0.01, $$

Article Snippet: Then, western blot analysis was performed as described previously.16,17 After blocking nonspecific binding with 5% BSA (room temperature, 1 h), membranes were consequently incubated with the primary antibody against KIM-1, RIPK1, RIPK3, p-MLKL, cleaved-caspase-3, cleaved-caspase-8 and β-actin overnight at 4 °C, followed by treated with IRDye 800-conjugated secondary antibody (Rockland immunochemicals, Gilbertsville, PA, USA).

Techniques: Western Blot, Real-time Polymerase Chain Reaction, In Vitro, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Wogonin protects against cisplatin-induced acute kidney injury by targeting RIPK1-mediated necroptosis.

doi: 10.1038/labinvest.2017.115

Figure Lengend Snippet: Figure 6 Wogonin fails to further reduce cisplatin-induced cell injury and inflammatory response in HK2 cells where RIPK3 is disrupted. (a) Western blot analysis and quantitative data of RIPK3 in HK2 cells. Results showed that RIPK3 was downregulated by transfection of RIPK3 shRNA plasmid. (b) Western blot analysis and quantitative data of RIPK1/RIPK3/MLKL signaling in RIPK3 knockdown HK2 cells. (c) Western blot analysis and quantitative data of KIM-1. Results indicated that when RIPK3 was knockdown, wogonin failed to further decrease the protein levels of KIM-1. (d) Real-time PCR identified that when RIPK3 was disrupted, wogonin failed to further decrease mRNA levels of TNF-α and IL-6. Independent experiments were performed throughout the in vitro studies in triplicate or quadruplicate. **Po0.01,***Po0.001 compared with the control. #Po0.05, ##Po0.01, ###Po0.001 compared with cisplatin-treated group. $$Po0.01,$$$Po0.001 compared with RIPK3 EV group. Cis, cisplatin; Wog, wogonin; EV, empty vector; KD, knockdown.

Article Snippet: Then, western blot analysis was performed as described previously.16,17 After blocking nonspecific binding with 5% BSA (room temperature, 1 h), membranes were consequently incubated with the primary antibody against KIM-1, RIPK1, RIPK3, p-MLKL, cleaved-caspase-3, cleaved-caspase-8 and β-actin overnight at 4 °C, followed by treated with IRDye 800-conjugated secondary antibody (Rockland immunochemicals, Gilbertsville, PA, USA).

Techniques: Western Blot, Transfection, shRNA, Plasmid Preparation, Knockdown, Real-time Polymerase Chain Reaction, In Vitro, Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Wogonin protects against cisplatin-induced acute kidney injury by targeting RIPK1-mediated necroptosis.

doi: 10.1038/labinvest.2017.115

Figure Lengend Snippet: Figure 9 Wogonin prevents RIPK1-mediated necroptosis in cisplatin nephropathy. (a) Electron microscope. Results clearly demonstrated that wogonin largely reduced nuclear structure damage and abnormal cell organelle content in injured kidney. (b) Western blot analysis of RIPK1/RIPK3/MLKL signaling. Results showed that treatment of wogonin significantly suppressed the activation of RIPK1/RIPK3/MLKL axis in cisplatin-injured kidney. Data represent the mean ± s.e.m. for 6–8 mice. ***Po0.001 compared with control. ##Po0.01, ###Po0.001 compared with model. Cis, cisplatin; Wog, wogonin.

Article Snippet: Then, western blot analysis was performed as described previously.16,17 After blocking nonspecific binding with 5% BSA (room temperature, 1 h), membranes were consequently incubated with the primary antibody against KIM-1, RIPK1, RIPK3, p-MLKL, cleaved-caspase-3, cleaved-caspase-8 and β-actin overnight at 4 °C, followed by treated with IRDye 800-conjugated secondary antibody (Rockland immunochemicals, Gilbertsville, PA, USA).

Techniques: Microscopy, Western Blot, Activation Assay, Control

Journal: International journal of molecular sciences

Article Title: Activation of the TNF-α-Necroptosis Pathway in Parvalbumin-Expressing Interneurons of the Anterior Cingulate Cortex Contributes to Neuropathic Pain.

doi: 10.3390/ijms242015454

Figure Lengend Snippet: Figure 2. TNF-α-necroptosis pathway is activated in the ACC of neuropathic pain models. Volcano map (A) and heat map (B) showing the expression of TNF-α, RIP1, RIP3, and MLKL in the ACC of CCI model of neuropathic pain (n = 3/group) and controls (n = 3/group) (data from GEO dataset GSE212311). The Hub gene expression data of CCI model (red dots) and control (green dots) for TNF-α, RIP1, RIP3, and MLKL are shown in (C). * p < 0.05 versus the control (unpaired t test). Volcano map (D) and heat map (E) showing the expression of TNF-α, IL-6, RIP1, and MLKL in the ACC of SNI model (n = 3/group) and controls (n = 3/group) (data from GEO dataset GSE228065). (F) Representative triple staining shows the overlap of TNF-α (green) with p-RIP3 (red) and PV (magenta), or p-MLKL (red) and PV (magenta) on PO day 7. Enlarged and color-split images of the area enclosed in white boxes are shown below. White arrows indicate co-localization. Blue fluorescence corresponds to DAPI.

Article Snippet: To seal it at room temperature, 5% skimmed milk was used to for almost 1 h, then end up placing the membranes in p-RIP1 antibody (1:1000, Affinity, Cincinnati, OH, USA), anti-p-RIP3 antibody (1:1000, CST, Danvers, MA, USA), anti-p-MLKL antibody (1:1000, Affinity, Cincinnati, OH, USA), anti-RIP1 antibody (1:1000, Novus, Littleton, CO, USA), anti-RIP3 antibody (1:1000, Novus, Littleton, CO, USA), anti-MLKL antibody (1:1000, Millipore, Billerica, MA, USA), and anti-TNF-α antibody (1:1000, Abcam, Cambridge, UK) overnight in the refrigerator at 4 ◦C.

Techniques: Expressing, Gene Expression, Control, Staining

Journal: International journal of molecular sciences

Article Title: Activation of the TNF-α-Necroptosis Pathway in Parvalbumin-Expressing Interneurons of the Anterior Cingulate Cortex Contributes to Neuropathic Pain.

doi: 10.3390/ijms242015454

Figure Lengend Snippet: Figure 3. SNI increases the expression of necroptosis-related protein p-RIP1 in the contralateral ACC (A) Representative Western blot of RIP1 and p-RIP1 expression in the bilateral ACC is shown in the top panel. The quantitative results of Western blotting protein are shown below. SNI induces the expression of p-RIP1in the contralateral ACC at postoperative (PO) day 7. ** p < 0.01 versus the sham group (two-way ANOVA). (B) Representative double staining shows the overlap (yellow) of p-RIP1 (red) with NeuN (neuronal marker, green) and PV (PV-IN marker, green), but not with GFAP (astrocyte marker, green) or Iba1 (microglia marker, green), on PO day 7. Enlarged and color- split images of the area enclosed in white boxes are shown in the middle. White arrows indicate co-localization (yellow). Blue fluorescence corresponds to DAPI. The fluorescence intensity curves for red and green from boxed areas are shown on the right side of each group.

Article Snippet: To seal it at room temperature, 5% skimmed milk was used to for almost 1 h, then end up placing the membranes in p-RIP1 antibody (1:1000, Affinity, Cincinnati, OH, USA), anti-p-RIP3 antibody (1:1000, CST, Danvers, MA, USA), anti-p-MLKL antibody (1:1000, Affinity, Cincinnati, OH, USA), anti-RIP1 antibody (1:1000, Novus, Littleton, CO, USA), anti-RIP3 antibody (1:1000, Novus, Littleton, CO, USA), anti-MLKL antibody (1:1000, Millipore, Billerica, MA, USA), and anti-TNF-α antibody (1:1000, Abcam, Cambridge, UK) overnight in the refrigerator at 4 ◦C.

Techniques: Expressing, Western Blot, Double Staining, Marker

Journal: Experimental Hematology & Oncology

Article Title: Tetrahydromagnolol targets TRIM38 to mediate PANoptosis in cancer cells and has the potential for synergistic cancer therapy

doi: 10.1186/s40164-025-00734-4

Figure Lengend Snippet: THM induced PANoptosis by assembling PANoptosome. A Immunoblot analysis was used to detect the activation markers of pyroptosis (GSDME, AIM2), apoptosis (PARP, Cleaved-CASP3, Cleaved-CASP8) and necroptosis (p-RIPK1, p-RIPK3, p-MLKL) in the cell lysates of HT29, HCT116, A549 and H1299 cells after treatment with THM. β-actin was used as a loading control. B Representative images of IHC staining for Ki67, Cleaved-PARP, Cleaved-CASP3, AIM2, p-MLKL and p-RIPK1 in tumor tissues, scale bar = 50 μm. C-D HCT116 (75 µM) and A549 (90 µM) cells were treated with THM for 24 h, then immunofluorescence confocal laser microscopy analysis was performed after staining with the specified PANoptosome complex antibodies. Representative images illustrated the pairwise co-localization of ASC with CASP8, RIPK3, and AIM2 respectively. Scale bar = 20 μm

Article Snippet: Primary antibodies were incubated with slides overnight at 4 °C, including Ki67 (CST, 9449, 1:1500), Cleaved-PARP (CST, 5625, 1:50), Cleaved-CASP3 (CST, 9664, 1:2000), AIM2 (Proteintech, 20590-1-AP, 1:100), p-MLKL (UpingBio, YP-Ab-10353, 1:200), p-RIPK1 (Proteintech, 28252-1-AP, 1:100) and TRIM38 (Proteintech, 13405-1-AP, 1:100).

Techniques: Western Blot, Activation Assay, Control, Immunohistochemistry, Immunofluorescence, Microscopy, Staining

Journal: The Pakistan Veterinary Journal

Article Title: Melatonin Modulates Necroptosis and Enhances Antioxidant Defense during PGF-Induced Luteal Regression in Heat-Exposed Rats

doi: 10.29261/pakvetj/2025.008

Figure Lengend Snippet: Fig. 6: Effects of heat exposure and melatonin administration on the protein expression of RIPK1, RIPK3, and their phosphorylation, in the rat ovaries following PGF injection. (A-C) Protein expression levels of RIPK1, p-RIPK1, and the p-RIPK1/RIPK1 ratio at 16h post -PGF; (D-F) Protein expression levels of RIPK1, p-RIPK1, and the p-RIPK1/RIPK1 ratio at 24h post-PGF. (G-I) Protein expression levels of RIPK3, p-RIPK3, and the p- RIPK3/RIPK3 ratio at 16h post-PGF; (M, N, O) Protein expression levels of RIPK3, p-RIPK3, and the p-RIPK3/RIPK3 ratio at 24h post-PGF. Results are presented as mean±SEM from four rats at each time point. Significant differences are represented by different letters (P<0.05).

Article Snippet: Sections were subjected to overnight incubation at 4°C with primary antibodies using RIPK1 Rabbit pAb (A7414, Abclonal) at dilution of 1:50 and RIPK3 Rabbit pAb (A5431, Abclonal) at dilution of 1:100.

Techniques: Expressing, Phospho-proteomics, Injection

Journal: The Pakistan Veterinary Journal

Article Title: Melatonin Modulates Necroptosis and Enhances Antioxidant Defense during PGF-Induced Luteal Regression in Heat-Exposed Rats

doi: 10.29261/pakvetj/2025.008

Figure Lengend Snippet: Fig. 7: Immunohistochemic al and the localization of RIPK1 and RIPK3 proteins in the corpus luteum of rats 16h post-PGF injection. (A): RIPK1, (B): RIPK3, among different groups. NC indicates negative control. The upper panel of the figure displays the results of IHC, illustrating protein expression in the CL, using scale bar of 50 and 20μm. The middle panel presents Hematoxylin, DAB staining and threshold. The lower panel presents the quantitative analysis of the score count of DAB staining using Image J. Data are presented as means±SEM from three rats per time point.

Article Snippet: Sections were subjected to overnight incubation at 4°C with primary antibodies using RIPK1 Rabbit pAb (A7414, Abclonal) at dilution of 1:50 and RIPK3 Rabbit pAb (A5431, Abclonal) at dilution of 1:100.

Techniques: Injection, Negative Control, Expressing, Staining

Journal: Scientific Reports

Article Title: Mitigating effects of Jiawei Chaihu Shugan decoction on necroptosis and inflammation of hippocampal neurons in epileptic mice

doi: 10.1038/s41598-025-89275-8

Figure Lengend Snippet: Western blot antibodies and dilution factors.

Article Snippet: Rabbit Anti-RIP1 (Proteintech 13176-1-AP) , 1:3000.

Techniques: Western Blot, Synthesized