Ab3325

Transfected primary chondrocytes were lysed with ice-cold lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40 with proteinase and phosphatase inhibitors). 50 μg of protein lysates were run on a 10% SDS-PAGE and transferred to a 0.45 μm PVDF-membrane by semi-dry transfer (PerfectBlue™, PeqLab). The membrane was blocked with 5% milk and incubated with the appropriate primary antibodies anti-GFP (1:15000, Abcam, #ab13970, RRID:AB_300798), anti-Myc (1:1000, Cell Signaling Technology, mAb #2276, RRID:AB_331783), pan-CaMKII (1:1000, Cell Signaling Technology, mAb #4436S, RRID:AB_10545451), anti-Flag (1:1000, Sigma-Aldrich, #F1804, RRID:AB_262044), and proteasome 20 (P20) (1:15000, Abcam, #ab3325, RRID:AB_303706), followed by incubation with the respective, species-specific HRP-coupled secondary antibodies (1:1000 and 1:5000). ECL substrate was used for signal development (Amersham, #RPN2106) on X-ray film (Amersham).

Antibodies used were against; USP4 (2651, Cell Signaling), AQP2 [16 (link)], Ser256 phosphorylated AQP2 [17 (link)], proteasome 20s (ab3325, Abcam), α-ENaC [18 (link)], actin (A2228, Sigma), AQP4 (AQP-004, Alomone, Jerusalem, Israel), V2R [19 (link)], Na-K-ATPase α1 subunit [20 (link)], H+-ATPase B1 subunit [21 (link)] and ubiquitin (P4D1, Cell Signaling). DUB inhibitor PR619 (Sigma), phosphatase inhibitor, and protease inhibitor (PhosSTOP and cOmplete mini tablets, Roche Diagnostics A/S) were added to all the isolation and wash buffers used in immunoprecipitation and biotinylation experiments.

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.