Vivaspin centrifugal concentrator

The truncated MtDXPS gene was obtained commercially as a synthetic gene and cloned into a pETM-11 plasmid using the NcoI and HindIII restriction sites. Expression, lysis and the initial IMAC purification were performed as above for full-length MtDXPS. The protein-containing fractions were subsequently combined and diluted with a low-salt buffer consisting of 50 mM HEPES pH 8.0, 5% glycerol, 5 mM dithiothreitol (DTT) and 100 µM MgCl₂ to a conductivity of 8 mS/cm. The solution was then loaded on a Resource Q anion exchange column and eluted with a linear NaCl gradient from 0 to 1 M. The protein-containing fractions were pooled and purified by gel filtration on a HiLoad 16/600 Superdex 200 pg column equilibrated with 20 mM HEPES pH 8.0, 250 mM NaCl, 5% glycerol, 5 mM DTT. The protein-containing fractions were concentrated to 5.5 mg/mL using a Vivaspin centrifugal concentrator (MWCO 30 kDa, Sartorius), and the His-tag was cleaved by TEV-protease digestion at 10 °C overnight. Removal of the tag and protease was achieved by reversed IMAC chromatography, and the protein was purified again by gel filtration on a HiLoad 16/600 Superdex 200 pg column using 20 mM MOPS pH 7.50, 200 mM NaCl, 5% Glycerol, 2 mM DTT as buffer. The purified protein was concentrated to 10 mg/mL in a Vivaspin centrifugal concentrator (MWCO 10 kDa, Sartorius).

Fresh first morning urine samples (50 mL) were collected in sterile containers and processed within 1 h after collection. Urinary cells and debris were removed by centrifugation at 2000× g for 30 min at 4 °C, obtaining the first fraction cell-free urine. Then, only 50 mL of the collected supernatant were transferred to clean tubes and centrifugated at 10,000× g for 30 min at 4 °C to eliminate large microvesicles. After this centrifugation, the urine supernatant was added to a Vivaspin centrifugal concentrator (Sartorius) and then centrifugated at 3000× g for 30 min, in order to concentrate urine proteins.
Human LOX-1 levels were assessed from human urine RCC (n = 30) and ctr (n = 25) with Human Lectin like Oxidized Low Density Lipoprotein Receptor1 (LOX-1) ELISA kit (MB52703808, Mybiosource, Inc. San Diego, CA, USA), in accordance with the manufacturer’s instructions. The absorbance was measured with the spectrophotometer Multimode detector DTX 880 (Beckman Coulter, Milan, Italy).

In order to create phosphorylated response regulator we adapted the method described by McCleary and Stock^{45 (link)}. There, response regulators (2–3 µg) were incubated with 50 mM acetyl phosphate for 30 min. The reactions were conducted at 37 °C in (25 mM Tris/HCl pH 7.8, 5 mM MgCl₂, 4 mM 2-mercaptoethanol, 5% ethylene glycol), followed by buffer exchange to (30 mM Tris/HCl, pH 8.0) with Vivaspin centrifugal concentrator (Sartorius, Germany). The final phosphoprotein concentrations were adjusted to 1.5 mg/ml (80 µM). Both kinase and phosphatase reactions were conducted in (25 mM Tris/HCl pH 7.8, 5 mM MgCl₂, 4 mM 2-mercaptoethanol, 5% ethylene glycol), where all the desired proteins (2–4 µg each) were incubated in 10 µl total volume at 25 °C, with or without 1 mM ATP. The reactions were started by adding ATP to the mixture and incubated either in dark or under saturating 657 nm red light. After 20–30 min, the reactions were stopped by adding 5× SDS loading buffer. For the mobility shift detection of phosphorylated RR proteins^{35 (link)}, we applied Zn²⁺-Phos-tag® SDS-PAGE assay (Wako Chemicals). The 9% SDS-PAGE gels containing 25-µM Phos-tag acrylamide were prepared, and 10 µl of each reaction were run at 40 mA/gel at room temperature according to manufacturer instructions. See Source Data for full gels.