Immunoblotting Analysis of NCC and Related Proteins

Protein samples were separated using 4–15% gradient polyacrylamide gels (Criterion TGX Precast Protein Gels, BioRad) and transferred electrophoretically to PVDF membranes. Immunoblotting was performed using standard methods and the following primary antibodies: rabbit polyclonal antibodies against NCC (SPC-402D, StressMarq), phosphorylated Threonine-58 NCC (pT58-NCC) (Pedersen et al., 2010 (link)), Nedd4-2 (Kamynina et al., 2001 (link)), phosphorylated Serine373 SPAK/Serine325 OSR1 (pS373-SPAK) (07–2273, Sigma), SPAK/OSR1 (Moriguchi et al., 2005 (link)), Kir4.1 (APC-035, Alomone), Kir5.1 (LS-C177333, LS Bio), Proteasome 20S (ab3325, Abcam) and Actin (A2066, Sigma) plus a mouse monoclonal NCC antibody (Kortenoeven et al., 2021 (link)). Blots were developed using SuperSignal West Femto chemiluminescent substrate (Thermo Scientific, Denmark) or Amersham ECL Western Blotting Detection Reagent (GE Healthcare) detection and the intensity of the bands were quantified using Image Studio Lite (Qiagen) densitometry analysis.

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Rosenbaek L.L., Petrillo F., van Bemmelen M.X., Staub O., Murali S.K, & Fenton R.A. (2022). The E3 ubiquitin-protein ligase Nedd4-2 regulates the sodium chloride cotransporter NCC but is not required for a potassium-induced reduction of NCC expression. Frontiers in Physiology, 13, 971251.

Publication 2022

Criterion TGX Precast Protein Gels APC-035 ab3325 SuperSignal West Femto chemiluminescent substrate Amersham ECL Western Blotting Detection Reagent Image Studio Lite

Actin Antibodies Densitometry Gels Membranes Monoclonal antibody Mouse Polyacrylamide gels Proteasome Protein Pvdf Rabbit Threonine

Corresponding Organization : Aarhus University

Other organizations : University of Lausanne, University of Veterinary Medicine Vienna

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Variable analysis

independent variables

Protein samples

dependent variables

Intensity of the protein bands

control variables

4–15% gradient polyacrylamide gels (Criterion TGX Precast Protein Gels, BioRad)
PVDF membranes
Standard immunoblotting methods
Primary antibodies: rabbit polyclonal antibodies against NCC, pT58-NCC, Nedd4-2, pS373-SPAK, SPAK/OSR1, Kir4.1, Kir5.1, Proteasome 20S, Actin, and a mouse monoclonal NCC antibody

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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