Complete protease inhibitor cocktail

At designated times, the treated cells were removed from the incubator and placed on ice. The cells were then washed 3 times with ice-cold PBS and lysed for 30 min with RIPA lysis buffer [50 mM Tris–HCl (pH 7.4), 1% Triton X-100, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 100 mM phenylmethylsulfonyl fluoride, 1 μg/ml of leupeptin, 1 mM Na3VO4, and 1× Complete Protease Inhibitor Cocktail (Santa Cruz Biotechnology)]. Equal amounts of protein were loaded onto 10–15% SDS-PAGE gels, electrophoresed, and transferred onto PVDF membranes (Millipore, Bedford, MA). The membranes were blocked in Tris-buffered saline with 0.05% Tween 20 (TBST) supplemented with 5% powdered milk, and then incubated with primary antibody against ADRP or β-actin. The blots were then washed with TBST and incubated with a horseradish peroxidase-conjugated secondary antibody in TBST plus a 5% solution of powdered milk. The bound antibodies were detected with Amersham ECL Prime Western Blotting Detection (GE Healthcare, Buckingharmshire, UK).

Whole cell protein lysates were prepared from the cell lines by using RIPA lysis buffer containing complete Protease Inhibitor Cocktail (Santa Cruz, CA, United States) according to the manufacturer’s protocols. Seventy five micrograms of whole cell protein were separated in a 3–8% NuPAGE Tris-Acetate gel, transferred to PVDF membrane (Thermo Fisher Scientific, Carlsbad, CA, United States), and then immunoblotted with the primary antibodies in 0.05% Tween 20-Tris-buffered saline (TBST) containing 5% skim milk at 4°C with shaking overnight. The primary antibodies used were: a mouse monoclonal anti-TET2a that recognizes the amino terminus of TET2, (C15200179, Diagenode) at a dilution 1:1,000 and a polyclonal rabbit-anti-TET2b (R1086, Abiocode) that recognizes the C-terminus of the short isoform of TET2, at 1:800. The β-actin, used as an internal control, was detected by rabbit-anti -β-actin polyclonal antibody (Cell Signaling Technology, MA, United States). HPR-conjugated anti-rabbit and mouseIgG (1:8,000, Cell Signaling Technology) and anti-rabbit IgG (1:3000, Cell Signaling Technology, MA, United States) were used as secondary antibodies. The membranes were incubated with SuperSignal West Pico peroxide and luminal enhancer solutions (Pierce, IL, United States) for 5 min, exposed to the film and developed.

MDCK cells stably expressing WT- and mutant V2R-GFP were grown to 80% confluence, and incubated with the growth medium containing 100 μM cycloheximide (CHX, Sigma-Aldrich, Milan, Italy) for 0, 4, or 8 h. The cells were subsequently lysed with the solution containing 1% NP-40, protease (Complete Protease Inhibitor Cocktail, Santa Cruz, USA) and phosphatase inhibitor cocktail (PhosSTOP, Roche, Monza, Italy), and immunoblots were performed as described above. Protein concentration was quantified using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA).

Cultured cells with or without treatment were lysed on ice in Lysis Buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na₃VO₄, 1 μg ml⁻¹ leupeptin, Cell Signaling Technology 9803) supplemented with Complete Protease Inhibitor Cocktail (Santa Cruz Biotechnology sc-29130) and phosphatase inhibitors (PhosSTOP, Roche 04906845001). For xenografted tumour samples, ∼1 g of the tumour tissue was ground in liquid nitrogen and transferred to a clean Eppendorf tube. The tissue pellets were resuspended in 600 μl of the Lysis Buffer, mixed gently on ice for 30 min and centrifuged at 14,000g for 10 min at 4 °C. The supernatant containing the whole-tumour tissue lysate was transferred to a fresh tube for further analyses. The protein concentrations of the lysates were measured using a BCA Assay kit (Thermo 23227). The whole-cell/tissue lysates were boiled with SDS–PAGE sample loading buffer, separated by SDS–PAGE, blotted on PVDF or nitrocellulose membranes and probed with the indicated antibodies. The signals were visualized using the Immobilon Western Chemiluminescent HRP Substrate (Millipore WBKLS0100). The uncropped blots for main figures were presented in Supplementary Fig. 42.

Human OFs were removed from the incubator and placed on ice. Cells were then washed three times with ice-cold PBS and lysed for 30 min with RIPA lysis buffer [50 mM Tris-HCl (pH 7.4), 1% Triton X-100, 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 100 mM phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 mM Na₃VO₄, and 1X Complete^™ Protease Inhibitor Cocktail (Santa Cruz Biotechnology)]. Equal amounts of protein extracts were loaded onto 10–15% SDS-PAGE gels, electrophoresed, and transferred to PVDF membranes (Millipore, Bedford, MA). Membranes were blocked in Tris-buffered saline with 0.05% Tween-20 (TBST) supplemented with 5% powdered milk or 5% bovine serum albumin, and then incubated with a primary antibody against the designated protein. Blots were then washed with TBST and incubated with a horseradish peroxidase-conjugated secondary antibody in TBST with 5% powdered milk. Bound antibodies were detected with the Amersham ECL Prime Western Blotting Detection kit (GE Healthcare, Buckinghamshire, UK).

Left kidneys were sectioned in Cortex/OSOM, ISOM and IM. Tissues were homogenized with a TissueLyser (Qiagen) in Lysis buffer (Sucrose 0,3M, Imidazole 25mM, EDTA 1mM, PMSF 1mM) with protease and phosphatase inhibitor cocktails (Complete Protease Inhibitor Cocktail, cod. Sc-29130, Santa Cruz; PhosSTOP, Roche). Total protein concentration was measured by Bradford assay (Biorad Protein Assay). SDS-PAGE was performed on NuPage 4–12% Bis-Tris Gel. Proteins were then transferred to PVDF membranes (Invitrolon PVDF, Invitrogen).
The membranes were probed with antibodies anti-Dicer1 (Dana-Farber Molecular Biology core Facilities, corresponding to residues 1385–1405) dilution 1:1000, anti-β-catenin (cat. no. 610153, BD Transduction Laboratories) dilution 1:1000, anti-AQP2 (dilution 1:1000), anti-NKCC2 (dilution 1:10000), anti-mTor (CST 2983; diluition 1:1000), anti Phospho-mTor (Ser2448) (CST 5536; diluition 1:1000) anti-S6 Ribosomal protein (CST 2317; diluition 1:1000), anti-PhosphoS6 Ribosomal protein (Ser235/236) (CST 2211; diluition 1:500), anti-β-Actin (Sigma, n. cat. T6793 dilution 1:20000) and GAPDH (GeneTex 100118; diluition 1:20000). Blots were incubated with HRP conjugated secondary antibodies, according to the species of the primary antibodies (Amersham) and then developed using ECL substrate (Pierce). Image-j was used to quantify single band intensity.

After rats (n = 24) were killed with isoflurane and pentobarbital, their brains were rapidly extracted and placed on ice. A 400-mg wedge of perilesion and lesion of injured cortex and striatum was removed, flash-frozen in liquid nitrogen and stored at -80°C. Prior to further extraction, frozen brain tissue was placed into prechilled Ambion RNAlater-ICE reagent (Ambion/Life Technologies, Austin, TX, USA) and kept at -20°C for a minimum of 16 hours in accordance with the manufacturer’s instructions. Tissue was homogenized on ice in PARIS Cell Disruption Buffer (Ambion) supplemented with a protease inhibitor (Complete Protease Inhibitor Cocktail; Santa Cruz Biotechnology, Santa Cruz, CA, USA) with Omni Tip probes (Omni International, Kennesaw, GA, USA). RNA and protein fractions were processed from the same starting tissue samples using the Ambion PARIS kit per the manufacturer’s instructions and stored at -80°C. RNA concentrations and integrity were determined by analysis on Experion RNA StdSens chips (Bio-Rad Laboratories, Hercules, CA, USA). Protein concentrations were determined by bicinchoninic acid assay (Thermo Scientific).