RIPK3 Antibody - #DF10141

Figure 2 Knockdown of HNEAP attenuates H/R‐induced cardiomyocyte necroptosis. a–d) The isolated neonatal mice cardiomyocytes were transfected with HNEAP agomir (HNEAP) or its negative control (NC) for 24 h. a) The expression level of HNEAP was analyzed by qPCR (n = 6 independent experiments). b) Necroptosis was determined by the PI staining. DAPI indicates Nucleus. Bar = 25 µm. c) Quantitative analysis of the percentage of necroptotic cells (n = 6 independent experiments). d) The activity of lactate dehydrogenase (LDH) in cardiomyocytes after transfection with HNEAP or NC. (n = 6 independent experiments). e) The isolated neonatal mice cardiomyocytes were transfected with HNEAP antagomir (anta) or its negative control (anta‐NC) for 24 h. The expression level of HNEAP was analyzed by qPCR (n = 4 independent experiments). f–i) The isolated neonatal mice cardiomyocytes were transfected with HNEAP antagomir (anta) or its negative control (anta‐NC) for 24 h and then cells were treated with H/R as described in the Experimental Section. f) qPCR analysis of the expression level of HNEAP (n = 5 independent experiments). g) Necroptosis was determined by the PI staining. DAPI indicates Nucleus. Bar = 25 µm. h) Quantitative analysis of the percentage of necroptotic cells (n = 6 independent experiments). i) The upper panel shows the activity of LDH in cardiomyocytes after transfection with anta or anta‐NC and treated with H/R (n = 6 independent experiments). The lower panel is representative western blot showing the expression of RIPK1 and RIPK3. Data are presented as Mean ± SD. All data were analyzed using one‐way ANOVA.

Figure 4 Restoration of NAD+ increases redox reactions and decreases apoptosis: a–c) Apoptosis in different groups of cells by detecting Annexin V/PI in flow cytometry (n = 3 independent samples; One-way ANOVA). d, e) Real-time PCR showing transcript levels of intracellular inflammatory and apoptosis indicators under different treatments (n = 3 independent samples; One-way ANOVA). f–i) Western blot data showing changes of expression of inflammatory- and apoptosis-related proteins under NMN supplemention and si-CD31 or NAT treatment (n = 3 independent samples; One-way ANOVA). GAPDH was used as loading control for RIPK1, RIPK3, Bcl-2, Caspase-3, BAX, SOD1, SOD2 and Caspase-9 in (g). β-actin was used as loading control for SOD1and SOD2 in (i). GAPDH was used as loading control for Bcl-2, Caspase-3, BAX, and Caspase-9 in (i). Data are shown as mean ± SEM. The P values are indicated on the graphs. NS, not significant.

Fig. 6 The anti-inflammatory effect of Fetuin-A is by inhibiting necroptosis in glutamate-exposed microglial cells. The microglial cells were treated with 100 μm glutamate (Glu) for 24 h to induce cellular injury. Meanwhile, Fetuin-A was treated into medium at indicated concentrations. A, B Expression of RIPK3, MLKL, p-RIPK3 and p-MLKL in microglial cells under various treatment conditions. GAPDH was used as the loading control. And bar graphs show the results of analysis (by band density analysis) of these proteins (n = 3). C–F Immunofluorescence assessment of p-RIPK3 and p-MLKL expression. Scale bar is 50 μm. The relative immunofluorescence intensity of Fetuin-A was detected with Image-J software (n = 3). G RIPK1 was immunoprecipitated with its antibody and resulted in co-immunoprecipitation of RIPK3. Immunoprecipitation of RIPK3 with its antibody caused co-immunoprecipitation of RIPK1 in microglial cells (n = 3). H Transmission electron microscopy (TEM) images of tissues. Translucent cytoplasm, mitochondrial swelling and destruction of membrane integrity were observed in TNF-α + zVAD-treated microglial cells (scale bar = 5 or 1 μm, n = 3). I IL-6, IL-1β, TNF-α, and IL-10 secretion was measured using ELISA (n = 5). Data are presented as the means ± SD; *P 

Figure 6. OTUB1 promotes the progression of BLCA depending on β-catenin/RIPK3/MLKL/necroptosis signaling pathway. A. Relative expression of OTUB1, β-catenin and downstream targets following overexpressed OTUB1 with or without XAV-939 (5uM, 10uM) treatment; relative expression of OTUB1, β-catenin and downstream targets following knockdown OTUB1 and/or overexpressed β-catenin. B. The RNA expression of OTUB1, β-catenin, RIPK3 and MLKL in elevated OTUB1, β-catenin groups with or without XAV-939 treatment. C, E, F. MTT assay, clone formation experiments and healing assay showed the proliferation ability of BLCA cell following overexpressed OTUB1 with or without XAV-939 (5uM, 10uM) treatment; coupled with that following knockdown OTUB1 and/or overexpressed β-catenin. D. Transwell assay showed the invasion ability of BLCA cells following overexpressed OTUB1 with or without XAV-939 (5uM, 10uM) treatment; coupled with that following knockdown OTUB1 and/or overexpressed β-catenin.

Fig. 2 Homer1-KD promoted post-ischemic neuronal necroptosis. A, B Representative images A and quantification B of neuronal death based on TUNEL assay in the ischemic penumbra cortex of each group of mice at 8 h after pMCAO. C Effects of Homer1-KD on the expression level of p-RIPK1, p-RIPK3, and p-MLKL in the ischemic penumbra cortex of each group at 8 h after pMCAO. D–G Quantification of result in C. H Representative photographs of p-MLKL-positive neurons of brain tissue in each group at 8 h after pMCAO. I Quantification of result in H. J, K Representative images J and quantification K of primary neuronal death based on TUNEL assay in vitro of different groups at 4 h after OGD. L Effects of Homer1-KD on the expression level of p-RIPK1, p-RIPK3, and p-MLKL of different groups in vitro experiment at 4 h after OGD. M–P Quantification of result in L. For B, D–G, I, K–N: *P