Trans blot turbo instrument

Kidneys were lysed in ice-cold extraction buffer at pH 7.5 (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.27 M sucrose, 0.1% β-mercaptoethanol and HALT protease and phosphatase inhibitor cocktail; Thermo Fisher Scientific). Tissue was homogenized, frozen in liquid nitrogen, immediately thawed and incubated at 4 °C on a nutator for 30 min and centrifuged at 13 000 r.p.m. for 5 min. Supernatant protein (100 μg) was combined with protein load buffer (100 mM Tris-HCl, pH 6.8, 200 mM DTT, 4% SDS, 0.2% bromophenol blue, 20% glycerol), heated at 95 °C for 5 min, loaded onto 4–20% precast SDS-PAGE gels (Bio-Rad, Hercules, CA, USA) and transferred to PVDF membrane using the Trans-blot Turbo Instrument (Bio-Rad). Membranes were blocked with 1% skim milk in TBS-T (Tris-buffered saline/0.05% Tween-20) and primary antibodies were added: rabbit anti-Nedd4-2,^{34 (link)} mouse anti-β-actin (clone AC15; Sigma-Aldrich), or ENaC and NCC antibodies described above. HRP secondary antibodies were added (Merck Millipore, Billerica, MA, USA) and developed with a ECL Prime substrate (GE Healthcare, Paramatta, NSW, Australia) on a ChemiDoc Touch Imager (Bio-Rad).

Following transfection, cells were treated with 50 μM MG132 (Boston Biochemical, Boston, MA, USA) and 400 μM chloroquine (Sigma‐Aldrich) for 4 h before lysis in Verhagan lysis buffer (1% triton X‐100, 10% glycerol, 150 mM NaCl, 20 mM Tris HCl pH 7.4 and 2 mM EDTA). For ubiquitination assays, 5 mM N‐ethylemaleimide (Sigma Aldrich) was added to the lysis buffer to inhibit protein deubiquitination. Immunoprecipitation was performed on protein G Sephrose beads with goat polyclonal anti‐GFP antibody. Samples were loaded onto 4%–20% precast polyacrylamide gels (Bio‐Rad Laboratories, Hercules, CA, USA) for separation by electrophoresis. Proteins were then transferred to polyvinylidene difluoride (PVDF) membrane using a Trans‐blot turbo instrument (Bio‐Rad). Membranes were then blocked with 5% skim milk and immunoblotted with primary antibody diluted in 5% skim milk in TBS‐T (Tris‐buffered saline with 0.05% Tween 20) incubated overnight at 4°C, followed by incubation with an alkaline phosphatase (AP), horseradish peroxidase (HRP) or Cy5‐conjugated secondary antibody. Visualization of HRP signals was using an ImageQuant LAS4000 (GE Healthcare) whereas a Typhoon FLA biomolecular imager (GE healthcare) was used to visualize signals from alkaline phosphatase and Cy5.

Western blot method was used to detect the levels of 2’, 3’ cyclic nucleotide 3’ phosphodiesterase (CNP). After protein extraction using RIBA buffer, samples were loaded on the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and were separated according to their molecular weights (BioRad mini protein electrophoresis separation unit and BioRad electrophoresis power supply). Following electrophoresis, proteins were transferred from the gel to Polyvinylidene difluoride (PVDF) membrane using BioRad Trans-Blot Turbo instrument. The membrane was blocked using 3% bovine serum albumin (BSA) in tris-buffered saline with 0.1% Tween 20 at room temperature for 1 hour. Afterwards, the membrane was incubated with a 1:2000 dilution of antibodies against CNP (Thermo Fisher, USA) at 4°C overnight. Next, membranes were probed with horseradish peroxidase-conjugated immunoglobulins solution (HRP-lmg Goat mab -Novus Biologicals) for 1 hour at room temperature. The chemiluminescent substrate (ClarityTM Western ECL substrate - BIO-RAD, USA cat#170-5060) was applied to the blot and signals were captured using a CCD camera-based imager. Image analysis software was used to read the band intensity of the target proteins against control sample after normalization by beta actin on the Chemi Doc MP imager.

Cells were harvested by removal of media and addition of RIPA buffer (Cell Signaling Technology #9806, diluted to 1X in deionized water) supplemented with 1X Halt Protease Inhibitor Cocktail (Thermo Scientific #78430) and incubated on ice for 30 minutes. Cell lysate was centrifuged at 21,000 × g for 10 minutes and supernatant was mixed with Blue Loading Buffer (Cell Signaling Technology #7722) per the manufacturer’s recommended ratio. The combined supernatant and loading buffer was boiled for 8 min at 97°C and cooled to room temperature. Samples were run on 4–20% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad #4561096) in Tris/Glycine/SDS buffer (Bio-Rad #1610732, diluted to 1X in deionized water). Transfers were performed using a Bio-Rad Trans-Blot Turbo instrument, RTA Mini 0.2 μm PVDF Transfer Kit (Bio-Rad #1704272), and Trans-Blot Turbo Transfer Buffer (Bio-Rad #10026938). CRX (A-9) (Santa Cruz Biotechnology #sc-377138) and beta-Tubulin Rabbit Ab (Cell Signaling Technology #2146S) were used, along with Anti-rabbit IgG, HRP-linked (Cell Signaling Technology #7074S) and Anti-mouse IgG, HRP-linked (Cell Signaling Technology #7076S). TBST (EZ BioResearch #S-1012, diluted to 1X in deionized water) and EveryBlot Blocking Buffer (Bio-Rad #12010020) were used for washing and blocking, respectively.