Complete and phosstop

Total protein from kidneys was extracted by homogenizing tissue in RIPA buffer (Thermo Fisher Scientific) containing protease and phosphatase inhibitors (PhosSTOP and Complete, Roche). Following incubation at 4°C and centrifugation, supernatant was collected and measured for protein concentration using a BCA assay (Pierce), aliquoted and stored at -80°C. Protein lysates (10 μg) were used to measure concentrations of pro Collagen, type 1 alpha 1 (Col1a1), utilizing an ELISA kit (ab210579, Abcam) according to the manufacturer’s instructions. Data from UUO kidneys were correlated to corresponding CL kidney to reduce variability between mice. Group average was calculated and expressed as fold change compared to WT CL kidney. For Western blot analysis, 20 μg denatured proteins were separated on 4–20% MiniProtean gels (BioRad) and then transferred to 0.2 μm PVDF membranes. Blots were blocked with 5% BSA and incubated overnight with primary antibody. After washing and incubation with the appropriate HRP-conjugated secondary antibody, proteins were visualized using ECLplus detection reagents (GE, Uppsala, Sweden). To ensure equal protein loading, the same blot was stripped with stripping buffer (L7710A, Interchim) and then incubated with an anti-GAPDH antibody.

For Native PAGE analysis of the oligomeric state of p63 two ovaries per indicated
condition were harvested in 20 µl of ice-cold lysis buffer A (50 mM Tris pH 8.0,
100 mM NaCl, 1 mM DTT, 2 mM MgCl₂, supplemented with 1x cOmplete and
PhosSTOP (Roche)). Lysis was performed by mechanical force using a pestle, pipetting
and two cycles of freeze and thaw. After addition of 20 µl lysis buffer B (lysis
buffer A containing 40 mM CHAPS) and 1 µl benzonase, samples were incubated for 1 hr
on ice and subsequently centrifuged for 10 min at 4°C and 13.2 krpm to remove cell
debris. 20 µl of supernatant were supplemented with 5 µl of 5x Native PAGE sample
buffer (60% glycerol, 25 mM coomassie G250) for Native PAGE analysis. The remaining
lysate was used for analysis of p63 level and phosphorylation-induced mobility shift
via SDS-PAGE.
The separation of ovary lysate by Native PAGE followed by detection of p63 via
subsequent Western Blot analysis was performed with the Native PAGE Novex 3–12%
Bis-Tris protein gel system (Life Technology) according to the manufacturer’s
instructions. The cathode buffer was supplemented with 0.002% coomassie G250 and the
separation was performed at 4°C for 60 min at 150 V and 90 min at 250 V.

Cells were pre‐treated for 2 h with 1 μg/ml of doxycycline (Clontech) and then 3 μM of asunaprevir (Asv; ApexBio) and/or 500 μM IAA were added to the media for indicated time periods. Cells were harvested and lysed in EBC buffer (50 mM Tris pH 7.5, 120 mM NaCl, 0.5% NP‐40, 1 mM EDTA, 1 mM DTT, protease and phosphatase inhibitors [Complete and PhosStop; Roche Diagnostics]) then mixed with 5× sample buffer (0.01% bromophenol blue, 62.5 mM Tris–HCl pH 6.8, 7% SDS, 20% sucrose and 5% β‐mercaptoethanol). The samples were sonicated then boiled at 95°C for 5 min. Samples were analysed by Western blotting and the signal was detected using Immobilon Western Chemiluminescent HRP substrate (Millipore). The intensity of cyclins A2 and B1 signals was quantified using ImageJ software. GAPDH was used to normalise the samples.

Cells were harvested in lysis buffer (200 mM ammonium bicarbonate, pH 7.5, 8 M urea) supplemented with the PhosStop phosphatase inhibitor cocktail Roche Diagnostics (Rotkreuz, Switzerland), reduced and alkylated, and then digested with Trypsin/LysC Promega (Madison WI, USA) after dilution to 2 M urea. The peptides were acidified to 1 % TFA (trifluoroacetic acid) and desalted on SepPak C18 cartridges. Lyophilized peptides were dissolved in immunoprecipitation buffer (50 mM ammonium bicarbonate, pH 7.4, 150 mM NaCl, 1 % (w/v) octyl-β-d-glucopyranoside, and Roche protease and phosphatase inhibitors (Complete and PhosStop, Roche Diagnostics, Rotkreuz, Switzerland) and incubated with 200 μl of anti-phosphotyrosine antibodies (PY99; SantaCruz, Dallas TX, USA) for 16 hours at 4 °C. After elution of the beads with 0.1 % TFA, peptides were desalted on Poros R3 and further purified on TiO₂ microcolumns.

Protein samples were prepared by homogenizing cardiac tissue in radioimmunoprecipitation assay (RIPA) buffer containing proteinase and phosphatase inhibitors (Complete and PhosSTOP; Roche, Basel, Switzerland). Protein extracts from myocardial tissue were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to 0.22 μm polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were then blocked in Tris-buffered saline with 0.1% Tween 20 containing 5% nonfat dry milk for 2 h at room temperature. Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies, including Nr4a1 recombinant rabbit monoclonal antibody (Cat#MA5-32647, Thermo Fisher Scientific, 1:1000) and HK2 recombinant rabbit monoclonal antibody (Cat#ab209847, Abcam, 1:1000;). The housekeeping protein GAPDH (Cat#KM9002T, Sungene Biotech, 1:4000) was used as reference protein to normalize the target proteins during western blot analysis. After washing, the membranes were incubated with secondary antibodies antibody IgG (Cat# A0208, Beyotime Biotech, 1:1000) at the appropriate dilutions for 1.5 h at room temperature, and detected using the enhanced chemiluminescence substrate kit. The density of each band was quantified using Quantity One. Three biological repeats were performed for each sample.

Cells for western blot experiments were replated the night before collection at sub confluent density, starved for 2 h for investigated cytokine, rinsed once with DMEM-F12 before treatment. Cells were lysed on ice with RIPA buffer containing protease and phosphatase inhibitors (Complete and PhosSTOP, Roche). Samples were centrifuged for 3 min at 15000 × g to remove cell debris and treated with DNAse to degrade genomic DNA. Protein concentration was determined by BCA reaction (Thermo) according to the manufacturer’s instructions. Equal amounts of protein (15–50 μg) were loaded in equal volumes to SDS-Polyacrylamide gradient gels. Proteins were transferred to nitrocellulose membranes and probed with indicated antibodies. The same membrane was stripped and probed with various antibodies unless otherwise stated. Details of antibodies are listed in Supplementary Table 3.