Protectin DX, LY294002 (PI3K inhibitor), and H89 (PKA inhibitor) were from Cayman Chemical Company (Ann Arbor, MI). LPS (Escherichia coli serotype 055:B5) was purchased from Sigma (St. Louis, MO). Interleukin-1 (IL-1), interleukin-10 (IL-10), tumor necrosis factor-α, myeloperoxidase, and cAMP ELISA kits were from R&D Systems (Minneapolis, MN). BOC-2 (ALX inhibitor), Rp-cAMP (cAMP inhibitor), and Rp-cGMP (cGMP inhibitor) were obtained from Biomol-Enzo Life Sciences (Farmingdale, NY). Anti-Na, K-ATPase α1 and β1 antibodies were purchased from Abcam (Cambridge, MA), and anti-sodium channel α, β, and γ antibodies were purchased from Biorbyt (Cambridge, Cambridgeshire). Anti-P-Akt, total Akt (T-Akt), and Nedd4–2 antibodies were obtained from Cell Signaling Technology (Beverly, MA).
Nedd4 2
Manufactured by Cell Signaling Technology
Nedd4-2 is an E3 ubiquitin-protein ligase that regulates the ubiquitination and subsequent degradation of various target proteins. It plays a role in the regulation of ion channels, transporters, and signaling proteins.
Automatically generated - may contain errors
Lab products found in correlation
Lps escherichia coli serotype 055 b5, by Merck Group (1 mentions)
Camp elisa kit, by R&D Systems (1 mentions)
Protectin dx, by Cayman Chemical (1 mentions)
Ly294002, by Cayman Chemical (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence, by Cytiva (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
P pka, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase, by Thermo Fisher Scientific (1 mentions)
Irs 1, by Cell Signaling Technology (1 mentions)
3 protocols using nedd4 2
1
Protectin DX Modulates Inflammatory Signaling
2
Western Blot Analysis of Kidney Proteins
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For Western blot analysis, kidney cortical lysates were prepared as previously described17 (link),48 (link). Kidney tissue samples weighing between 15–25 mg were pulse sonicated in Laemmli buffer with a protease inhibitor cocktail (Roche, Basel Switzerland) to a concentration of 20 mg/ml. The resulting supernatant was subjected to SDS-PAGE, transferred onto nitrocellulose membrane (Millipore, Bedford MA), probed with antibodies and subsequently visualized by enhanced chemiluminescence (Amersham Biosciences Inc, Piscataway, NJ). For Western blots, we used the following antibodies: α-, β-, γ-ENaC, and NKCC2 antibodies (1:1000, Stressmarq Biosciences Inc, Vancouver, cat# SPC-403D, SPC-404D, SPC-405D, and SPC-401D, respectively), NCC antibody (1:1000, kindly provided by Dr. David Ellison, Oregon Health and Sciences University), α-NKA antibody (1:2000, Santa Cruz Biotech cat# sc-365091), and Nedd4-2 and pNedd4-2 (1:1000, Cell Signaling cat# 4013 S and 8063S, respectively). Western blot densitometry quantification was determined using Image Lab software (BioRad, Hercules, CA) and normalized to actin loading controls.
3
Immunoblotting with Antibody Cocktail
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The following primary antibodies were used for Western blotting, Nedd4-2, pPKA consensus sequence and IRS1 (Cell Signalling). Anti-rabbit and anti-mouse secondary antibodies conjugated to horseradish peroxidase (HRP) were from Thermo Fisher Scientific (Rockford, IL, USA) and GE Healthcare (Little Chalfont, UK), respectively.
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