Cells were seeded into six-well plates at a density of 2 × 105 cells per well and grown overnight. Cells in wells were transfected with 1000 ng of plasmids including pDCUg-NT, pDCUg-FL, pDM-NT and pDM-FL, respectively. At 48 h post transfection, the whole-cell extracts were prepared using a Total protein Extraction kit (BC3711, Solarbio, China) according to the manufacturer’s instructions. The protein lysates (20 μg/sample) were resolved by SDS-PAGE and the target proteins were detected with Western blot (WB) using the antibodies as follows: GAPDH Rabbit monoclonal antibody (ab181602, Abcam, UK) (1:10,000 dilution), SLC40A1 Rabbit polyclonal antibody (ab58695, Abcam, UK) (1:10,000 dilution), and Lipocalin-2 Rabbit polyclonal antibody (ab63929, Abcam, UK) (1:10,000 dilution). The second antibodies were IRDye® 800CW Goat anti-Rabbit IgG (C80118-05, Licor) (1:10,000 dilution). The blots were detected and fluorescence intensity was quantified with the Odyssey Infrared Fluorescence Imaging System (Licor) and Odyssey software.
Ab58695
Manufactured by Abcam
Sourced in United Kingdom
Ab58695 is a laboratory product manufactured by Abcam. It is a tool used for research purposes. No further details about its core function or intended use can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Ab236773, by Abcam (2 mentions)
Ab48394, by Abcam (2 mentions)
Ab2302, by Abcam (2 mentions)
Ab63929, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Odyssey infrared fluorescence imaging system, by LI COR (1 mentions)
Irdye 800cw goat anti rabbit igg, by LI COR (1 mentions)
Total protein extraction kit, by Solarbio (1 mentions)
Imagequant las 500, by GE Healthcare (1 mentions)
P0012, by Beyotime (1 mentions)
Ab73895, by Abcam (1 mentions)
Goat anti rabbit igg conjugated to hrp, by Bio-Rad (1 mentions)
Nbp1 21502, by Novus Biologicals (1 mentions)
Ab8227, by Abcam (1 mentions)
Ab1086, by Abcam (1 mentions)
Ab55735, by Abcam (1 mentions)
Gel imaging analysis system, by Analytik Jena (1 mentions)
5 protocols using ab58695
1
Protein Expression Analysis by Western Blot
2
Iron Homeostasis and Apoptosis in Epileptogenesis
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As previously described (Zhang M. et al., 2020 (link)), the cortex and hippocampus of each rat were isolated on days 3 and 21 during epileptogenesis. Proteins were extracted using a reagent kit (P0012, Beyotime, China). Rabbit monoclonal antibodies against ferroportin 1 (FPN1, 1:2000, ab58695, Abcam, Cambridge, UK), iron regulatory protein 1 (IRP1, 1:1000, ab236773, Abcam, UK), microtubule-associated protein light chain 3beta (LC3B, 1:2000; ab48394, Abcam, UK), caspase-3 (1:1000, 9662, Cell Signaling Technology, Boston, MA, USA), activated caspase-3 (1:1000, ab2302, Abcam, UK), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:3000, AB-P-R001, Kangcheng, Nanjing, China) were used for western blot analysis. Strip images were acquired using an image analyzer (ImageQuant LAS 500, GE Healthcare, Uppsala, Sweden). Grayscale analysis of target strips was performed using ImageJ V.1.37 software (National Institutes of Health, Bethesda, MD, USA).
3
Antibody-based Protein Expression Analysis
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All antibodies were commercially produced, tested, and recommended for the application. The primary capture antibodies in the ELISA analyses were rabbit anti-human ferroportin peptide (ab58695; Abcam, Cambridge, UK), rabbit anti-human IRP2 (LS-C80347, LifeSpan Biosciences, Seattle, WA, USA), rabbit anti-human HIF2 (ab73895; Abcam, Cambridge, UK). Mouse anti-human Immunoglobin G (IgG) conjugated with horseradish peroxidase (HRP; #55788; BD, Franklin Lakes, NJ, USA) was used as secondary capture antibody. The primary antibody used in the Western blot analyses was a rabbit anti-human ferroportin (NBP1-21502; Novus Biologicals, Littleton, MA, USA). A rabbit anti-human β-actin antibody (ab8227; Abcam, Cambridge, UK) was used as loading control. For Western blot analyses, the secondary antibody was goat anti-rabbit IgG conjugated to HRP (Bio-Rad, Sundbyberg, Sweden). Primary and secondary antibodies were used at 1 μg/mL.
4
Quantitative Analysis of Iron Transport Proteins
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Three iron transport-related proteins (DMT-1, TfR-1, and FP1) were detected by Western blotting. Frozen heart tissue samples were homogenized with 400 μL lysis buffer/20 mg tissue and then centrifuged at 12,000 r/min for 10 min, after which the supernatant was collected. Total proteins were loaded and separated on a 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel and then transferred to a nitrocellulose membrane. Blocking was performed with 5% skimmed milk powder in phosphate buffer saline (PBS), and then the membranes were incubated with primary antibodies overnight at 4°C. The membrane was washed three times (10 min each) to remove uncombined primary antibodies, and then it was incubated with secondary antibodies at room temperature for 90 min. After the membrane was washed three times to remove unbound secondary antibodies, the membrane was exposed and scanned by the Gel imaging analysis system (UVP, USA), and the gray value was automatically measured by the system. The primary antibodies included anti-DMT1 antibody (ab55735, Abcam), anti-transferrin receptor antibody [MEM-189] (ab1086, Abcam), and anti-ferroportin/SLC40A1 antibody (ab58695, Abcam).
5
Hypoxia-Induced Protein Expression Analysis
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As previously described [2 (link)], at 24 h, 3 days, and 2 months after hypoxia, we extracted proteins with a commercial extraction reagent kit (Beyotime Institute of Biotechnology, China). Anti-rabbit FPN1 (1:2000, ab58695, UK), IRP1 (1:1000, ab236773, Abcam, UK), LC3B (1:2000; ab48394, Abcam, UK), caspase-3 (1:1000, 9662, Cell Signaling Technology, USA), activated caspase-3 (1:1000, ab2302, Abcam, UK), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:3000; AB-P-R001, Kangcheng, Zhejiang, China) antibodies were used for western blotting analysis, based on chemiluminescent luminescent image analysis (ImageQuant LAS 500, USA). Grayscale analysis was performed on the target band using Image J (version 1.37, National Institutes of Health, Bethesda, MD), and the results are expressed as a ratio of the optical density of the target protein band to that of GAPDH.
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