Tim23 Antibody - #DF12052

Fig. 1 Morphine induced NLRX1-mediated mitophagy in microglia. A The mitochondrial DNA (mtDNA) levels were decreased significantly in primary microglia with 1.0 μM morphine as measured by mtDNA/nDNA analysis (n = 4). B Co-staining of HSP60 or Tim23 and LC3B in primary microglia. Bar = 10 μm. C The quantified results of AD-mCherry-GFP-LC3B transfection in BV2 cells with morphine treatment (n > 50 cells). The Kruskal–Wallis test was employed to indicate statistical significance. The results of mCherry-dots are shown in red and the mCherry-GFP-dots are shown in yellow. D Representative Western blots of BV2 cells in control or morphine treatment group (n = 3–6). E The expression of NLRX1 mRNA was peaked in primary microglia with 1.0 μM morphine as measured by qPCR (n = 3). One-way ANOVA were employed in A, E. F Quantitative graphs of NLRX1 mRNA in BV2 cells (n = 6). G, H Confocal microscopy analysis of NLRX1 (red), LC3B (green) and DAPI (light blue). Bar = 2 μm. The Pearson’s correlation coefficients of NLRX1 and LC3B were elevated in morphine-treated BV2 cells (H, n = 6–9 fields). I Co-immunoprecipitation analysis of NLRX1 and LC3 in BV2 cells (n = 3). J, K Western blots analysis of NLRX1-mediated mitophagy in BV2 cells by siRNA (J, n = 5) or shRNA (K, n = 3). L The quantified results of AD-mCherry-GFP-LC3B transfection in BV2 cells with NC-siRNA or NLRX1-siRNA pre-treatment (n > 50 cells). Data represent the mean ± SEM. Student's t-test or Mann–Whitney U test were used to measure significance between two groups. (* p < 0.05, ** p < 0.01, *** p < 0.001 and ns p > 0.05)

Fig. 2 NLRX1-mediated mitophagy occurred in microglia independent of PINK1–Parkin pathway, but not astrocyte (A, B) Quantitative graphs of NLRX1 mRNA (A, n = 6) and representative Western blots of mitophagy (B, n = 3) in primary astrocyte. Quantitative graphs of NLRX1 mRNA (C, n = 6) and representative Western blots of mitophagy (D, n = 3) in MA cells. E Co-staining of HSP60 or Tim23 and LC3B in primary astrocyte. Bar = 10 μm. F The quantitative graphs of JC-1 assays as measured by flow cytometry (n = 4). G The NLRX1 mRNA expression of BV2 cells with or without CCCP treatment (n = 6). Data represent the mean ± SEM. Two-sided Student’s t tests were used to measure significance between two groups (* p < 0.05, ** p < 0.01, *** p < 0.001 and ns p > 0.05)