This was performed as previously described (Huebner et al. 2016 (link)). Briefly, 10 μg protein per sample was analyzed. Primary antibodies for iron homeostatic proteins were used in a 1:2000 dilution against FTN heavy chain (sc-25617, Santa Cruz Biotechnology, Dallas TX), transferrin (TF sc-30159, Santa Cruz Biotechnology), transferrin receptor-1 (TFRc 13-6890, Life Technologies, Rockville MD), and GAPDH (1:5000, G8795; Sigma-Aldrich, St Louis MO). Immunoreactive bands were visualized utilizing isotype-specific IR-dye conjugated secondary antibodies (A-21109, Life Technologies; #926-68022, 1:15,000, LI-COR, Lincoln, NB; #610-132-007, 1:30,000, Rockland Immunochemicals, Gilbertville, PA). Band IR intensities were quantified using the Odyssey infrared imaging system and Image Studio software (both from LI-COR). Molecular weights for bands of interest were calculated against color-tagged protein standards (Precision Plus Protein, BioRad, Hercules CA). Protein band intensity was normalized to GAPDH expression within the same sample and protein abundance was compared between samples; PAE does not affect GAPDH abundance in this model, as determined by comparison to total protein loaded per lane (Huebner et al. 2016 (link)). Samples were assayed in triplicate.
Sc 25617
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-25617 is a lab equipment product offered by Santa Cruz Biotechnology. It is a scientific instrument designed for use in various laboratory applications. The core function of this product is to facilitate specific research and analysis procedures, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Sc 7396, by Santa Cruz Biotechnology (2 mentions)
Precision plus protein, by Bio-Rad (1 mentions)
A21109, by Thermo Fisher Scientific (1 mentions)
G8795, by Merck Group (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Image studio software, by LI COR (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Sc 30203, by Santa Cruz Biotechnology (1 mentions)
Mouse anti gapdh nb300 221, by Novus Biologicals (1 mentions)
Sc 371, by Santa Cruz Biotechnology (1 mentions)
Anti ha probe f 7 sc 7392, by Santa Cruz Biotechnology (1 mentions)
Sc 101713, by Santa Cruz Biotechnology (1 mentions)
Ecl western blotting detection system, by Bio-Rad (1 mentions)
Neuronal nuclei (neun), by Cell Signaling Technology (1 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Ecl western blotting detection system, by Santa Cruz Biotechnology (1 mentions)
Anti γ tubulin antibody c 20, by Santa Cruz Biotechnology (1 mentions)
7 protocols using sc 25617
1
Quantifying Iron Homeostasis Protein Levels
2
Western Blot Analysis of Alkaline Phosphatase and Ferritin
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Western blotting was performed with 10% SDS‐PAGE gels and 0.45 μm pore size nitrocellulose membrane (Amersham Biosciences, Little Chalfont, UK). Alkaline phosphatase was detected with a rabbit polyclonal antibody at 1:200 dilution (sc‐30203; Santa Cruz Biotechnology, Dallas, TX, USA) followed by HRP‐labelled anti‐rabbit IgG secondary antibody 1:15,000 (Amersham Biosciences). Ferritin heavy chain was detected using primary antibody against human FtH at 1:400 dilution (sc‐25617; Santa Cruz Biotechnology) and secondary antimouse IgG antibody (Amersham Biosciences) was used at 1:15,000 dilution. Antigen–antibody complexes were visualized with the horseradish peroxidase chemiluminescence system (Amersham Biosciences). Membranes were reprobed for glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). Mouse anti‐GAPDH (NB300‐221; Novus Biologicals, Littleton, CO, USA) at 1:1000 and secondary antimouse IgG (Amersham Biosciences) at 1:15,000 dilution were used.
3
Western Blot Analysis of Protein Expression
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A total of 40 µg protein extract was boiled for 10 min in SDS sample buffer, separated by 12% SDS-PAGE and transferred to a nitrocellulose membrane by electroblotting. Non-specific reactivity was blocked in non-fat dry milk in Tween-PBS (5% (w/v) milk in phosphate-buffer saline (PBS) (pH 7.4) and 0.005% Tween 20) for 2 h at room temperature. The nitrocellulose membranes were incubated overnight at 4 °C with the following antibodies: (a) anti-FHC (H-53) (1:200; sc-25617, Santa Cruz Biotechnology, Dallas, TX, USA), (b) anti-p65 (C-20) (sc-372, 1:1000; Santa Cruz Biotechnology), (c) anti-HDAC (AV38530, 1:5000; Sigma-Aldrich), (d) anti-HA probe (F-7) (sc-7392, 1:1000; Santa Cruz Biotechnology), (e) anti-γ-Tubulin antibody (C-20) (1:3000; sc-7396, Santa Cruz Biotechnology), (f) anti-phospho-IκBα (Ser 32/36) (1:1000; sc-101713, Santa Cruz Biotechnology), and (g) anti-IκBα (C-21) (1:1000; sc371, Santa Cruz Biotechnology).
Membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies and immunoreactive bands were visualized with the ECL Western blotting detection system (BioRad, Hercules, CA, USA).
Membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies and immunoreactive bands were visualized with the ECL Western blotting detection system (BioRad, Hercules, CA, USA).
4
Quantifying FHC Expression in Prelimbic Neurons
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Imaging and analysis was performed as previously described (Pitcher et al., 2013 (link)), with modifications. Briefly, fluorescent microscopy coupled with multispectral image analysis was used to identify individual neurons immunostained for NeuN (Cell Signaling Technology 24307, 1:400) within layer 2/3 of the mPFC prelimbic region. Within those neurons, the average fluorescent signal of FHC [H-53] immunostaining (Santa Cruz Biotechnology sc-25617, 1:100), a semi-quantitative measure of FHC expression, was measured. FHC average fluorescent signals were measured from at least 1000 layer 2/3 neurons in two separate slices from each animal, and these values were averaged to one value for each animal before statistical analysis. Immunohistochemical staining, image acquisition, and analysis were each performed by different people, and two people both blinded to the treatment condition separately performed analysis.
5
FHC Protein Quantification in K562 Cells
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Whole-cell lysis, protein extraction and Western Blot analyses of cultured K562shScr, K562shFHC, K562cntr, and K562siFHC cells were performed, as previously reported [25 (link),26 (link)]. For FHC protein quantification, the incubation of the anti-rabbit polyclonal primary anti-FHC (H-53) (1:200; sc-25617, Santa Cruz Biotechnology, Dallas, TX, USA) followed by the incubation with the HRP-conjugated secondary antibody (1:3000 Cell Signaling) was carried out. The goat polyclonal anti-γ-Tubulin antibody (C-20) (1:3000; sc-7396, Santa Cruz Biotechnology) was used as loading control. The immunoreactive bands were visualized with the ECL Western blotting detection system (Santa Cruz Biotechnology) and the bands intensity was quantified by using ImageJ (National Institutes of Health, Bethesda, MD, USA).
6
Western Blot Analysis of Iron Metabolism Proteins
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Caco-2 cells and THP-1 cells were lysed in 300 μL of 25 mM MOPS pH 7.4, 150 mM NaCl, 1% Triton, 1 mM PMSF, 2 μM leupeptin and pepstatin in ice for 1 h. Total protein content was quantified by Bradford assay. An amount of 20 μg of total protein, in SDS sample buffer containing DTT, was heat-treated (except for Fpn [17 (link)]) and loaded onto SDS-PAGE. For Western blot analysis, the following primary antibodies were employed: monoclonal anti-TfR1 (anti-TfR) (sc-32272, Santa Cruz, CA, USA) (1:5000), monoclonal anti-Fpn 31A5, (Amgen) (1:10,000), polyclonal anti-Ftn (sc25617, Santa Cruz, CA, USA) (1:10,000), polyclonal anti-HCP (A0031, Dako, Santa Clara, CA, USA) (1:10,000), anti-hephaestin (sc-365365, Santa Cruz, CA, USA) (1:10,000), anti-DMT-1 (sc-166884, Santa Cruz, CA, USA) (1:10,000) and monoclonal anti-actin (sc1616, Santa Cruz, CA, USA) (1:10,000). After incubation with the appropriate secondary horseradish peroxidase-conjugated antibody, blots were developed with Enhanced ChemiLuminescence (ECL Prime) (GE Healthcare, UK). Protein levels were normalized on actin by densitometry analysis, performed with ImageJ.
7
Quantifying Intracellular Iron in Dermal Fibroblasts
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Quantitation of intracellular iron concentration in dermal fibroblasts was performed as described by Reimer et al. [44 (link)]. Briefly, cells were lysed using 50 mM NaOH for 2 h, neutralized by addition of equal volume of 10 mM HCl and sonicated. Protein concentration was determined using the DC protein assay, and lysates were then treated with freshly prepared acidic KMnO4 solution (1.4M HCl and 4.5% (w/v) KMnO4 in H2O) at 60°C for 2 h with shaking. The total iron content of the lysates was determined by the addition of an iron detection reagent (6.5 mM ferrozine, 1 M ascorbic acid, 2.5 M ammonium acetate) followed by measurement of absorbance at 550 nm. To measure the expression of ferritin in fibroblasts, a rabbit polyclonal anti-ferritin heavy chain antibody (Santa-Cruz Biotechnology, sc-25617) was used.
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