mitofusin2 d2d10 rabbit mab catalogue no 9482  (Cell Signaling Technology Inc)

Journal: Journal of Pharmaceutical Analysis

Article Title: Naringenin boosts Parkin-mediated mitophagy via estrogen receptor alpha to maintain mitochondrial quality control and heal diabetic foot ulcer

doi: 10.1016/j.jpha.2025.101333

Figure Lengend Snippet: Naringenin (NAR) maintains mitochondrial homeostasis under high glucose (HG) stress. (A) HaCaT cells exposed to HG (50 mM) for 24 h were treated with NAR (0.12, 0.37, 1.1, and 3.3 μM) for 24 h. The expression levels of mitochondrial function-related proteins, including adenosine triphosphate (ATP) synthase F1 subunit alpha (ATP5F1A), cytochrome C oxidase subunit 2 (MT-CO2), and nicotinamide adenine dinucleotide (NADH) dehydrogenase (ubiquinone) iron-sulfur protein 4 (Ndufs4), in HaCaT cells were measured by Western blotting. (B) HaCaT cells were incubated with MitoSOX probes for 30 min to detect mitochondrial superoxide levels, followed by inverted fluorescence microscope. (C) The expression levels of mitochondrial biogenesis-related proteins, including peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), nuclear respiratory factor 1 (NRF1), heat shock protein 60 (HSP60), transcription factor A, mitochondrial (TFAM), translocase of inner mitochondrial membrane 23 (Tim23), cytochrome C oxidase subunit 4 (COX IV), and translocase of outer mitochondrial membrane 20 (Tomm20), in HaCaT cells were measured by Western blotting. (D) The messenger RNA (mRNA) levels of PGC-1α, TFAM, and NRF1 were measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). (E) The protein levels of mitofusin 2 (MFN2) and dynamin-1-like protein (DRP1) in HaCaT cells were determined by Western blotting. The arrowhead indicates the band of MFN2. (F) Representative fluorescence images of mitochondrial morphology of HaCaT cells stained with MitoTracker as observed by confocal microscope and two-dimensional (2D) threshold images using ImageJ. (G, H) Analysis of the mean mitochondrial area (G) and mean form factor (FF) (H) from F using ImageJ. Data are presented as mean ± standard deviation (SD). ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001, compared with the control group. GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: After blocking with 5% fat-free milk in Tris-buffered saline containing 0.1% Tween 20, the PVDF membrane was incubated with primary antibodies against β-actin (Santa Cruz Biotechnology), β-tubulin (Biodragon, Suzhou, China), p-H2A histone family member X (p-γH2AX) (Bioss, Beijing, China), adenosine triphosphate (ATP) synthase F1 subunit alpha (ATP5F1A) (Sangon Biotech Co., Ltd.), Cav-1 (Sangon Biotech Co., Ltd.), cytochrome C oxidase subunit 4 (COX IV) (Proteintech), dynamin-1-like protein (DRP1) (Sangon Biotech Co., Ltd.), ERα (Proteintech), ERβ (Sangon Biotech Co., Ltd.), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Proteintech), glutaredoxin 1 (GRX1) (Proteintech), histone H3 (Proteintech), heat shock protein 60 (HSP60) (Sangon Biotech Co., Ltd.), LaminB1 (Sangon Biotech Co., Ltd.), LC3 (MBL), mitofusin 2 (MFN2) (Proteintech), cytochrome C oxidase subunit 2 (MT-CO2) (Sangon Biotech Co., Ltd.), nicotinamide adenine dinucleotide (NADH) dehydrogenase (ubiquinone) iron-sulfur protein 4 (Ndufs4) (Sangon Biotech Co., Ltd.), nuclear factor-κB (NF-κB) (Proteintech), NQO1 (Sangon Biotech Co., Ltd.), nuclear respiratory factor 1 (NRF1) (Proteintech), cyclin-dependent kinase inhibitor 1A (P21) (Proteintech), sequestosome-1 (P62) (Proteintech), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) (Proteintech), PTEN-induced putative kinase 1 (PINK1) (Novus Biologicals, Littleton, CO, USA), p-NF-κB (S536) (Cell Signaling Technology), p-PINK1 (S228) (Thermo Fisher Scientific Inc.), succinate dehydrogenase complex flavoprotein subunit A (SDHA) (Proteintech), superoxide dismutase 1 (SOD1) (Proteintech), SOD2 (Proteintech), transcription factor A mitochondrial (TFAM) (Proteintech), translocase of inner mitochondrial membrane 23 (Tim23) (BD Biosciences, San Jose, CA, USA), Tomm20 (Proteintech), and voltage-dependent anion-selective channel protein 1 (VDAC1) (Sangon Biotech Co., Ltd.) overnight at 4 °C.

Techniques: Expressing, Western Blot, Incubation, Fluorescence, Microscopy, Membrane, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Staining, Standard Deviation, Control