BNIP3 Antibody - #DF8188

Figure 7 BA activates autophagy and mitophagy via enhancing AMPK-mTOR -TFEB activity. (A) Western blotting for AMPK, p-AMPK, mTOR and p-mTOR expression levels, and TFEB nuclear translocation in the Sham, SCI and BA groups. The gels were run under the same experimental conditions, and the cropped blots are shown here. (B) The optical density values of the AMPK, p-AMPK, mTOR, p-mTOR, normalized to the loading control GAPDH; Densitometric analysis of TFEB, normalized to the loading control H3. (C) Western blotting for AMPK, p-AMPK, mTOR and p-mTOR expression levels, and TFEB nuclear translocation in the BA and BA+3MA groups. The gels were run under the same experimental conditions, and the cropped blots are shown here. (D) The optical density values of the AMPK, p-AMPK, mTOR, p-mTOR, normalized to the loading control GAPDH; Densitometric analysis of TFEB, normalized to the loading control H3. (E) Western blotting for Caspase-1, NLRP3, GSDMD, p62, LC3II, Bnip3, Nix and Parkin expression levels in the BA and BA+CC groups. The gels were run under the same experimental conditions, and the cropped blots are shown here. (F) The optical density values of the Caspase-1, NLRP3, GSDMD, p62, LC3II, Bnip3, Nix and Parkin expression levels, normalized to the loading control GAPDH. The values are expressed as the means ± SEM, n=5 per group. *p< 0.05 and **p< 0.01, vs. Sham group. #p< 0.05 and ##p< 0.01, vs. SCI group. &p< 0.05 and &&p< 0.01, vs. BA group.

Figure 6 BA attenuates mitophagy and reduces ROS accumulation after SCI. (A) ELISA of 8-OHdG, AOPP, and MDA in spinal cord lesions from Sham, SCI, BA and BA+3MA groups as indicated. (B) Immunofluorescence staining for Nix and NeuN co-localization in the spinal cords of the Sham, SCI, BA and BA+3MA groups (scale bar = 25 µm). (C) The quantitative mean optical density of the Nix in motor neurons of spinal cord lesion in each group. (D) Western blotting for Bnip3, Nix and Parkin expression levels in the Sham, SCI and BA groups. The gels were run under the same experimental conditions, and the cropped blots are shown here. (E) The optical density values of the Bnip3, Nix and Parkin expression levels were quantified and analyzed in the three groups. (F) Western blotting for Bnip3, Nix and Parkin expression levels in the BA and BA+3MA groups. The gels were run under the same experimental conditions, and the cropped blots are shown here. (G) The optical density values of the Bnip3, Nix and Parkin expression levels were quantified and analyzed in the both groups. The values are expressed as the means ± SEM, n=5 per group. *p< 0.05 and **p< 0.01, vs. Sham group. #p< 0.05 and ##p< 0.01, vs. SCI group. &p< 0.05 and &&p< 0.01, vs. BA group.

Fig. 9. Effects of SG formula on predicted targets expression of HFD induced - NASH model. (A-F) Representative western blot images and bar graphs of the relative expressions of BNIP3, BNIP3L, LC3BI, LC3BII and p62 in each group. * , * *, and * ** represent P 

Fig. 5. Changes in related protein levels in grass carp gill fed with different protein levels and exposed to nitrite for 96 h. Data represent the means of each group. Treatment 1 of the control group was used as a reference. NS: not significant.

Figure 6 Bexarotene promotes mitophagy and decreases ROS levels after SCI. (A) Immunofluorescence staining for GSDMD (pyroptosis-related marker, green), C-CASP1 (pyroptosis-related marker, red), NIX (mitophagy-related marker, red) and DHE (indicating ROS-positive cells, red) in neurons in the spinal cord (original magnification 30×). Scale bar: 25 μm. (B–E) Quantitative analysis of levels of GSDMD (B), C-CASP1 (C), NIX (D) and DHE (E) in A. (F–H) The levels of 8-OHdG and AOPP in the spinal cord were detected by ELISA, and the levels of MDA were detected by the thiobarbituric acid assay. (I, L) Western blot assay for pyroptosis-related and mitophagy-related proteins. Data were normalized to GAPDH. (M) The levels of mitophagy-related genes in the spinal cord were detected by qPCR and normalized to β-actin. Data are expressed as the mean ± SEM (n = 6 mice per group). *P < 0.05 and **P < 0.01, vs. SCI group; #P < 0.05 and ##P < 0.01, vs. SCI + Bex group (one-way analysis of variance with the least significance difference post hoc test). ASC: Apoptosis-associated speck-like protein containing a CARD; Bex: bexarotene; BNIP3: BCL2/adenovirus E1B 19 kDa interacting protein 3; C-CASP-1: cleaved Caspase 1; DAPI: 4′,6-diamidino-2-phenylindole; DHE: dihydroethidium; ELISA: enzyme-linked immunosorbent assay; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSDMD-N: gasdermin D-N; IOD: integrated optical density; MDA: malondialdehyde; NIX/BNIP3L: BCL2/adenovirus E1B 19 kDa interacting protein 3 like; NLRP3: NOD-like receptor thermal protein domain associated protein 3; SCI: spinal cord injury.

Figure 6 Bexarotene promotes mitophagy and decreases ROS levels after SCI. (A) Immunofluorescence staining for GSDMD (pyroptosis-related marker, green), C-CASP1 (pyroptosis-related marker, red), NIX (mitophagy-related marker, red) and DHE (indicating ROS-positive cells, red) in neurons in the spinal cord (original magnification 30×). Scale bar: 25 μm. (B–E) Quantitative analysis of levels of GSDMD (B), C-CASP1 (C), NIX (D) and DHE (E) in A. (F–H) The levels of 8-OHdG and AOPP in the spinal cord were detected by ELISA, and the levels of MDA were detected by the thiobarbituric acid assay. (I, L) Western blot assay for pyroptosis-related and mitophagy-related proteins. Data were normalized to GAPDH. (M) The levels of mitophagy-related genes in the spinal cord were detected by qPCR and normalized to β-actin. Data are expressed as the mean ± SEM (n = 6 mice per group). *P < 0.05 and **P < 0.01, vs. SCI group; #P < 0.05 and ##P < 0.01, vs. SCI + Bex group (one-way analysis of variance with the least significance difference post hoc test). ASC: Apoptosis-associated speck-like protein containing a CARD; Bex: bexarotene; BNIP3: BCL2/adenovirus E1B 19 kDa interacting protein 3; C-CASP-1: cleaved Caspase 1; DAPI: 4′,6-diamidino-2-phenylindole; DHE: dihydroethidium; ELISA: enzyme-linked immunosorbent assay; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSDMD-N: gasdermin D-N; IOD: integrated optical density; MDA: malondialdehyde; NIX/BNIP3L: BCL2/adenovirus E1B 19 kDa interacting protein 3 like; NLRP3: NOD-like receptor thermal protein domain associated protein 3; SCI: spinal cord injury.

FIGURE 3 FG‐4592 inhibits apoptosis in bone marrow stromal cell (BMSC) post‐oxygen–glucose deprivation (OGD) through activating HIF‐1α/BNIP3‐dependent autophagy. (A, B) Apoptosis measured by flow cytometry (FCM), and statistics were displayed as mean ± SD. (C–E) Western blot images and relative quantification of apoptosis‐related proteins (Bcl‐2, Bax) and Gapdh (n = 4). (F–K) Expression of HIF‐1α, BNIP3, P62, Beclin‐1, LC3, and Gapdh in BMSCs was detected by western blot. And relative quantification of HIF‐1α, BNIP3, P62, Beclin‐1, LC3 (n = 4). * means p value <0.05, ** means p value <0.01, and *** means p value <0.001; n.s. means no significance, p values were calculated with one‐way ANOVA, followed by Bonferroni's multiple comparison test