Three or more 7-wk old WT and BK5.EP4 mice were topically treated with 200 μl acetone or 2.5 μg TPA in 200 μl acetone, and total protein was isolated from the epidermis at 3, 12, and 24 h after treatment as previously described (Sung et al., 2006 (link)). Fifty to 75 μg epidermal lysates were separated on either an 8% or 15% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membranes (Thermo Scientific). Primary antibodies used were anti-MMP-9 (1:1000; Chemicon) for the 24 h samples, anti-pErk1/2 (1:1000), anti-Erk1/2 (1:1000), anti-pStat3 (1:1000), and anti-Stat3 (1:1000), all from Cell Signaling, Danvers, MA, for the 3 h samples, and anti-actin-HRP (1:1000; Santa Cruz Biotechnology) for all samples as loading controls. Antibodies against CYP1A1 and CYP1B1 (1:500, Santa Cruz Biotechnology) and aromatase (1:1000; Invitrogen, Carlsbad, CA) were also used. Secondary antibodies were anti-mouse-HRP (1:2500) and anti-rabbit-HRP (1:10000; GE Healthcare). Blots were blocked in 5% BSA in Tris-buffered saline containing 0.1% Tween 20 (TBST), primary antibodies were incubated overnight in 5% BSA in TBST at 4°C, and the secondary antibodies were incubated for an hour in 5% non-fat milk in TBST at room temperature. The membranes were washed, and SuperSignal West Femto Maximum Sensitivity Substrate (Pierce Biotechnology, Inc., IL) was used to detect antibody binding.
Aromatase
Manufactured by Thermo Fisher Scientific
Sourced in United States
Aromatase is a key enzyme involved in the biosynthesis of estrogens from androgens. It catalyzes the final and rate-limiting step in estrogen production.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride membrane, by Thermo Fisher Scientific (1 mentions)
Anti mouse hrp, by GE Healthcare (1 mentions)
Anti p stat3, by Cell Signaling Technology (1 mentions)
Anti rabbit hrp, by GE Healthcare (1 mentions)
Anti stat3, by Cell Signaling Technology (1 mentions)
Anti mmp 9, by Merck Group (1 mentions)
Anti actin hrp, by Santa Cruz Biotechnology (1 mentions)
Anti erk1 2, by Cell Signaling Technology (1 mentions)
Anti p erk1 2, by Cell Signaling Technology (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Gapdh clone 0411, by Santa Cruz Biotechnology (1 mentions)
Donkey anti rabbit igg, by Cytiva (1 mentions)
Goat anti mouse igg, by Promega (1 mentions)
2 protocols using aromatase
1
Topical TPA Induces Epidermal Protein Changes
2
Quantitative Immunoblotting of Human Fibroblasts
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Whole-cell protein lysates (harvested in RIPA buffer) or cytoplasmic fractions from primary human dermal fibroblasts were resolved using 10% SDS PAGE and transferred to a nitrocellulose membrane (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Following blocking with 5% non-fat dry milk, membranes were incubated with the following primary antibodies: FN (clone EP5, Santa Cruz Biotechnology, Santa Cruz, CA, USA), FN (clone F14, Abcam, Waltham, MA, USA), alpha tubulin (clone DM1A, Abcam, Waltham, MA, USA), Col IIIA1 (Proteintech, Rosemont, IL, USA), Col VA1 (clone C-5, Santa Cruz Biotechnology, Santa Cruz, CA, USA), aromatase (Invitrogen, Waltham, MA, USA), and GAPDH (clone 0411, Santa Cruz Biotechnology, Santa Cruz, CA, USA). This was followed by incubation with horseradish peroxidase-conjugated secondary antibodies: goat anti-mouse IgG (Promega, Madison, WI, USA) and donkey anti-rabbit IgG (Cytiva, Malborough, MA, USA). After washing, immunoblots were developed with chemiluminescence reagents according to the manufacturer's protocol (Pierce, Rockford, IL, USA). Signal intensities were quantified by densitometry, normalized against GAPDH or alpha tubulin in each sample, and expressed as the magnitude of increase compared with controls. Densitometry was calculated using Image J v.1.53t [59] .
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