For ferroportin analysis, non-reduced samples were used; other samples were reduced. Western blots were probed with antibodies to Phospho-Smad-1-5-8 (Cell Signaling), total Smad-1 (Cell signaling), Smad-5, Phospho-STAT3 (Cell Signaling), STAT3 (Cell Signaling), hepcidin (Fitzgerald), ferroportin (Novus Biologicals), GAPDH (Fitzgerald), or β-actin (Abcam).
Ferroportin
Manufactured by Novus Biologicals
Sourced in United States
Ferroportin is a protein involved in the cellular export of iron. It plays a crucial role in regulating iron homeostasis within the body.
Automatically generated - may contain errors
Lab products found in correlation
Stat3, by Cell Signaling Technology (3 mentions)
β actin, by Abcam (2 mentions)
P stat3, by Cell Signaling Technology (2 mentions)
Transferrin receptor1, by Thermo Fisher Scientific (2 mentions)
β actin, by Merck Group (2 mentions)
Oxphos antibody cocktail, by Abcam (2 mentions)
Ferritin, by Cell Signaling Technology (2 mentions)
Phospho smad1 5 8, by Cell Signaling Technology (1 mentions)
Total smad1, by Cell Signaling Technology (1 mentions)
Smad5, by Cell Signaling Technology (1 mentions)
Phospho stat3, by Cell Signaling Technology (1 mentions)
Gapdh, by Biosynth (1 mentions)
Hepcidin, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Gel documentation system, by Bio-Rad (1 mentions)
Bca kit, by Beyotime (1 mentions)
Protease and phosphatase inhibitor, by Beyotime (1 mentions)
Bcl2 associated x (bax), by Abcam (1 mentions)
Vegfa, by Abcam (1 mentions)
Erk1 2, by Abcam (1 mentions)
8 protocols using ferroportin
1
Western Blot Analysis of Iron Regulation
2
Quantitative Protein Expression Analysis
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For quantification of specific protein expression of iron handling targets the automated, gel-free Western blot system, Simple Western™ was used (Protein Simple, San Jose, CA, USA). The antibodies used were actin (catalog #3700S) and HO-1 (catalog #70081S) from Cell Signaling Technologies (Danvers, MA, USA); transferrin (catalog #NB500-418), ferroportin (catalog #NBP1-21502), and transferrin receptor (catalog #NBP2-32945) from Novus Biologicals (Centennial, CO, USA); DMT (catalog #PA5-35136) from Thermo Fisher Scientific (Waltham, MA, USA); and hepcidin (catalog #SAB1405228) from Sigma-Aldrich (St. Louis, MO, USA).
3
Western Blot Analysis of Mouse Heart Proteins
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Mouse heart tissue/cells were lysed for 30 min with RIPA buffer (Beyotime, China) supplemented with protease and phosphatase inhibitors (Beyotime, China) on ice. The lysate was centrifuged at 12 000 rpm for 15 min at 4 °C, and the protein concentrations of the supernatant were analyzed with a BCA kit (Beyotime, China). Each sample (20 µg of protein) was separated by SDS‒PAGE gels and transferred to PVDF membranes. The membranes were immersed in 5% milk in TBST buffer for 1 h at room temperature, followed by incubation with primary antibodies against β‐actin (CST, 1:1000), bax (Abcam, 1:1000), caspase3 (CST, 1:1000), caspase 8 (CST, 1:1000), Cyt‐c (Proteintech, 1:1000), GPx‐4 (Abcam, 1:1000), Bcl‐2 (Affinity, 1:1000), TGF‐β (CST, 1:1000), COX‐2 (Affinity, 1:1000), VEGF A (CST, 1:1000), FAK (CST, 1:1000), ERK1/2 (CST, 1:1000), p38 (CST, 1:1000), transferrin receptor (Abcam, 1:1000), and ferroportin (Novus, 1:1000) at 4 °C overnight. Then, the membranes were washed three times with TBST, followed by incubation with secondary antibodies (1:10 000) for 1 h at room temperature. The bands were visualized by using a gel documentation system (Bio‐Rad, USA) and quantified by ImageJ software.
4
Muscle Protein Oxidation and Iron Regulation
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Frozen gastrocnemius samples were lysed in RIPA lysis buffer (150 mM of NaCl, 50 mM of Tris‐HCl, 0.5% sodium deoxycholate, 1% Triton X‐100, 0.1% SDS, and 1 mM of EDTA) supplemented with protease and phosphatase inhibitor cocktail (Roche). Protein concentration was determined using BCA assay (Thermo Fisher Scientific). Fifteen or thirty micrograms of protein from cell or gastrocnemius lysates, respectively, were loaded per well on Mini‐Protean TGX Stain‐Free precast polyacrylamide gels (Bio‐rad) for SDS‐PAGE. Stain‐Free imaging was performed using Chemidoc MP imager in order to visualize total protein patterns. Proteins were then transferred onto PVDF membranes prior to immunoblotting analysis. Blots were probed with the following primary antibodies: P‐Thr172‐AMPK (Cell Signaling 2535), Total‐AMPK (Cell Signaling 2532), Ferritin (Sigma F5012), IRP2(PA‐116544), Transferrin Receptor 1 (Santa Cruz sc65882), NCOA4 (Santa Cruz C‐4), OxPhos cocktail (Thermofisher 8199), Ferroportin (Novus NBP1‐21502), Vinculin (Cell Signaling 4650). Protein carbonylation was assessed by measuring the levels of carbonyl groups using the OxyBlot protein oxidation detection kit (Sigma‐Aldrich S7150). Quantification analysis of blots was performed with Image Lab software (BioRad).
5
Western Blot Analysis of Iron Homeostasis
6
Western Blot Analysis of Iron Homeostasis
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Cells were lysed with NP-40 lysis buffer (25 mM tris (pH 7.4), 1% Triton X-100, 1% SDS, 1% sodium deoxy-cholate, 150 mM NaCl, aprotinin (2 μg/ml), 1 mM phenylmethylsulfonyl fluoride) containing complete protease inhibitor cocktail (Roche Diagnostics). Samples were separated by SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes before being incubated with primary and secondary antibodies. Bands were detected using chemiluminescence (Thermo Scientific). Membranes were probed with antibodies to β-actin (Sigma, Cat# A3854 ), ferroportin (Novus Biologicals, cat# NBP121502 ), transferrin receptor1 (Invitrogen, cat# 13-6890), IRP2 (Santa Cruz, cat# sc-33682), ferritin H (17 (link)), p-STAT3 (cell Signaling, cat# 9131S) and STAT3 (cell Signaling, cat#4904S ). Western blots were quantified using ImageJ software.
7
Western Blot Protein Analysis Protocol
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Lysates were generated as previously described (45 (link)). In brief, lysates were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto nitrocellulose membranes, and probed overnight at 4°C with antibodies against GFP (66002-1-Ig), SDHB (10620-1-AP), FECH (14466-1-AP), or Actin (66009-1) were purchased from Proteintech. Other antibodies used were, Ferroportin (21502, Novus), Ferritin (3998 Cell Signaling, Danvers, MA), and OXPHOS antibody cocktail (Abcam, ab110413). All antibodies were used at a 1:1000 dilution.
8
Western Blot Protein Analysis Protocol
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