Cystatin C Antibody - #DF6457

Fig. 7. Effects of Fundc1 regulation on NET formation and endothelial mitochondrial quality control. A. Intestinal CitH3-DNA and MPO-DNA complexes were assayed using ELISAs. B–C. Protein expression levels of Ly6G and CitH3 were measured by Western blot analysis. D-E. Intestinal CD31-positive endothelial cells were isolated with CD31-coated magnetic beads. Mitochondrial (MitoTracker) and lysosomal (LysoTracker) immunofluorescence colocalization was performed to explore the mitophagy level. F-G. Endothelial mitophagy parameters (p62, LC3 II/I ratio and mito-LC3 II) and a mitochondrial protein (Tim23) were examined using western blotting. The relative grayscale values were measured using ImageJ. H–I. Cytc levels in the cytosol and isolated mitochondria were examined by western blotting. J-K. Quantification of cytoplasmic and mitochondrial ROS in the intestinal endothelium was conducted using DCFH‐DA and MitoSOX Red probes. L. Mitochondrial membrane potential changes in endothelial cells were detected by JC-1 staining. M-N. Key proteins of mitochondrial fusion (Mfn1 and Opa1) and fission (Mff and Drp1) were examined by a Western blot assay. O–P. Immunofluorescence confocal imaging of mitochondrial (Tom20, green) and Drp1 (red) colocalization was used to evaluate mitochondrial fission. AAV, adeno-associated virus; NC, negative control. Data are shown as the means ± SD, ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

FIGURE 4 Increased NET levels inhibited mitophagy activation and aggravated Cytc leakage in the intestine. (A,B). Western blotting experiments to assess mitophagy flux (p62, LC3 II/I ratio and Mito-LC3 II), mitochondrial protein (TIM23) levels in human intestinal tissues. (C,D) The colocalization of LC3 and mitochondria (Tom20) in the intestine in WT and Pad4ΔPMN mice. Colocalization events of mitochondria and LC3 puncta are indicated in yellow. Scale bars = 100 μm. (E,F). Qualitative analysis of the number of mitophagosomes in intestinal epithelial cells via TEM. Scale bars = 5 μm. (G,H) Western blots were used to analyse the mitophagy parameters (p62, LC3 II/I ratio and Mito-LC3 II), mitochondrial biomarkers (Tim23) in animals. (I,J) The Cytc contents in the cytosol and in isolated mitochondria were examined by western blotting. Data are displayed as the mean values with SD. ns, no significance. *p 

Figure 4. The effects of MEL on the PGC-1α/TFAM pathway in GA tissues of middle-aged mice a-c. Western blot and qRT-PCR were applied to detect the effects of MEL on PGC-1α/TFAM pathwayrelated protein and mRNA levels, including cytochrome c oxidase subunit 4 (COX4), cystatin C (CYTC), nuclear respiratory factor 1 (NRF-1), mitochondrial transcription factor A (TFAM), p-P38, P38, and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), n=3. Data are manifested as mean ± SD * P