Sc 21713

Homogenized renal tissue was provided for particular fraction preparation by 3-step centrifugation as previously described [13 (link)]. After electrophoretic separation of proteins (20 µg per each lane) in 12% sodium dodecyl sulfate-polyacrylamide gel, the samples were transferred to a nitrocellulose membrane. The membrane was incubated overnight with primary antibodies against individual subunits of Na,K-ATPase (α1, 1:250, A-277, Sigma-Aldrich, St. Louis, MO, USA; β1, 1:200, sc-21713, Santa Cruz Biotechnology, Dallas, TX, USA), and subsequently incubated for 1.5 h with a horseradish peroxidase-linked secondary anti-mouse antibody (1:1000, #7076C, Cell Signaling Technology, Denver, CO, USA). For protein visualization, an enhanced luminol-based chemiluminescence was applied. The quantification of the relevant bands was assessed densitometrically using Image J software, and subsequently normalized to β-actin (1:1000, ab6276, Abcam, Cambridge, UK) serving as a loading control [13 (link)].

Primary antibodies of mouse anti-Na⁺/K⁺-ATPase A1/C464.6: sc-21712, Santa-Cruz (1:200), mouse anti-Na⁺/K⁺-ATPase β1 (C464.8): sc-21713, Santa-Cruz (1:200), mouse anti-GAPDH/sc-32233, Santa-Cruz (1:10,000) and mouse Anti-β-Actin, Clone AC-74 (A2228), Sigma (1:20,000) were used.
Secondary antibodies of donkey anti-mouse IgG (H + L) SA1-100/Invitrogen (1:10,000) and donkey anti-rabbit IgG (H + L) 31,458/Invitrogen (1:10,000) were used.

Cochleae were dissected from saline-perfused, formalin-fixed temporal bones of young adult female M. fascicularis (3 years old, N = 3) and decalcified with 0.1 M EDTA/phosphate buffer at 4°C for at least 6 weeks, then embedded in paraffin. The 5 μm sections were rehydrated, pretreated with sodium citrate buffer, then blocked in PBS containing 5% FBS, 1% BSA, and 0.05% Tween-20. The primary antibodies used in this study were rabbit antiserum against SLC12A2, generated using the amino-terminal region of the protein as an immunogen, and thus able to detect both the exon 21-included and -skipped isoforms (Abcam: ab59791, 1:100), mouse monoclonal antibody against ATP1B1 (Santa Cruz Biotechnology: sc21713, 1:400), and goat antiserum against KCNQ1 (Santa Cruz Biotechnology: sc10646, 1:200). Sections were incubated with primary antibodies at 4°C overnight, and signals were visualized using appropriate secondary antibodies, conjugated with either Alexa 488 or 568, followed by counterstaining with DAPI. Images were captured by fluorescence (DM2500, Leica Microsystems) and confocal (Axio 700) microscopy.