Na4P2O7

Populations of LNCaP/MyrHA-Akt1 or LNCaP/LacZ cells in log-phase growth were rinsed twice with phosphate-buffered saline (PBS), resuspended in Buffer M [50 mM HEPES pH 7.4, 10 mM NaCl, 5 mM MgCl₂, 0.1 mM EDTA plus protease inhibitor cocktail (Complete Mini, Roche Applied Science), 1 mM Na₃VO₄, 1 mM NaF and 1 mM Na₄P₂O₇.10 dH₂O] and homogenized at 4°C using a Potter-Elvehjem tissue grinder (12 strokes, 1800 rpm). 10⁷-10⁸ cells were used for each extraction method. High-speed (16,000 × g) pellets from either the conventional or modified approach were subjected to successive detergent extraction essentially as described [19 (link),21 (link),24 (link)]. Briefly, pellets were resuspended in Buffer A [25 mM 2-(N-morpholino)-ethanesulfonic acid, 150 mM NaCl, pH 6.5] and samples combined with an equal volume of Buffer A containing 2% Triton X-100, and phosphatase and protease inhibitors. Samples were incubated on ice for 60 min, centrifuged at 16,000 × g for 20 min at 4°C and supernatants collected as Triton-soluble (TS) material. Pellets were rinsed briefly with Buffer A and resuspended in Buffer B [10 mM Tris-Cl, pH 7.6, 150 mM NaCl, 60 mM β-octylglucoside and phosphatase and protease inhibitors]. Samples were incubated on ice for 30 min, centrifuged at 16,000 × g for 20 min at 4°C and supernatants collected as Triton-insoluble (TI) material.

The properties of the bottom sediment, which have been analysed, are as follows: particle size fractions, pH and redox potential. The analytical methods, as well as the above parameters, have been described in our previous studies (Tarnawski and Baran 2018 (link)). The content of total organic carbon (TOC) in sediments was determined using a CNS analyser (Vario EL Cube, Elementar Analysensysteme 2013). The content of humus compounds was extracted from bottom sediments using a mixture of 0.1 mol dm⁻³ Na₄P₂O₇ solution and 0.1 mol dm⁻³ NaOH (Mierzwa-Hersztek et al. 2018 ). The carbon of humic acids (Cha) was isolated in the extract of sodium pyrophosphate and a sodium base, whereas the carbon of fulvic acids (Ckf) was calculated from the difference between the amount of carbon (C ext) as well as the amount of humic acid carbon (Cha) in the extract. The extraction residue—non-hydrolysing carbon (Cnh)—was computed from the difference between the total organic carbon content (TOC) and the amount of carbon in the extract. In the prepared solutions of humic acids, light absorbance was measured at the 465 and 665 nm wavelength and the colour ratio (E₄/E₆) was computed (Mierzwa-Hersztek et al. 2018 ). In order to determine the dissolved organic carbon (DOC), the sediment samples were extracted in sediment: water ratio 1:10 v/v, by shaking on a rotary shaker for 24 h. Next, the samples were centrifuged in 50-ml tubes at 3000×g for 10 min, and filtered through a 0.45 μm membrane filter (Akkanen et al. 2005 (link)). The DOC content was measured using TOC analyser 1200 (Thermo Elektron).

P10 Adam23^Δ1/Δ1 and age-matched WT control mice were culled, and sciatic nerves were harvested into Eppendorf tubes, snap-frozen in liquid nitrogen, and stored at −80°C until further processing. Nerves were homogenized using a Sample Grinding Kit and homogenization buffer (25 mM HEPES, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1 mM Na₃VO₄, 2.5 mM Na₄P₂O₇, 20 mM NaF, 2.5 mM β-Glycerophosphate, 1% NP40, 1% Tx100, 1% SDS, 1% Deoxycholate, PMSF, PIM). Samples were then centrifuged at 4°C for 10 min at 20,000 × g, and supernatants were transferred into fresh tubes. Protein concentration was measured using the Pierce Protein Assay (BCA). Samples of appropriate, equalized protein concentrations and volumes were mixed with LDS Sample Buffer and frozen at −20°C or immediately used in WB.

iTRAQ labeling with two 8-plex iTRAQ kits was performed in the Proteomic facility of the Institute of Biomedicine of Seville using their standard protocols. Individual liver samples were isolated in urea lysis buffer (8 M urea, 25 mM Tris, 100 mM NaCl, 25 mM NaF, 10 mM Na₄P₂O₇, 50 mM β-glycerophospate, 1 mM Na₃VO₄, 1:100 protease inhibitors, and 1:100 deacetylase inhibitors, pH 8). Samples were sonicated for 10 s and centrifuged at 20,000 × g for 15 min at 4 °C. The supernatant was harvested and stored at −80 °C. Then, samples were reduced with 50 mM tris-(2-carboxyethyl)phosphine (AB Sciex) at 60 °C for 1 h with shaking and were subsequently alkylated using 200 mM S-methyl methanethiosulfonate (AB Sciex) for 30 min at room temperature. Samples were then trypsinized at 37 °C in a 10:1 ratio (w/w) of substrate/enzyme in a water bath overnight (Promega). Then, samples were speed-vac dried. The iTRAQ-labeling assay was conducted according to the manufacturer’s instructions (iTRAQ 8-plex, AB Sciex). Briefly, peptides were reconstituted in 1 M triethylammonium bicarbonate (Sigma-Aldrich St. Louis, MO, USA), 0.05% SDS, 1:100 phosphatase inhibitor cocktail, 1:100 protease inhibitor cocktail, and 0.002% benzonase (Novagen, Argentina) and labeled with one isobaric amine-reactive tag. After 2 h of incubation, labeled samples were pooled, dried at 45 °C, and stored overnight at 4 °C. iTRAQ-labeled samples were desalted using Oasis HLB C18 cartridges (Waters, Milford, MA, USA) and dried using a vacuum centrifuge. Peptides were then prefractionated using MCX Oasis columns (Waters) and increasing concentrations (50–2000 mM) of ammonium formate. Fractions were collected, individually washed using C18 cartridges, and dried. Peptides from each fraction were separated using nano-liquid chromatography (nano LC 1000, Thermo Scientific) and analyzed by means of nano-electrospray ionization (Proxeon Biosystems, Odense, Denmark) connected to a Q Exactive Plus Orbitrap mass spectrometer (Thermo Scientific). Briefly, 13 µl of each fraction was loaded, preconcentrated, and washed in an Acclaim PepMap (75 µm × 2 cm, nanoViper, C18, 3 µm, 100 Å) precolumn (Thermo Scientific). Peptides were separated in an analytical column (75 µm × 15 cm, nanoViper, C18, 2 µm, 100 Å, Acclaim PepMap RSLC) for 240 min at 200 nL/minute (Thermo Scientific). Peptides were eluted with a gradient of buffer A (0.1% formic acid, 100% H₂O) to buffer B (0.1% formic acid, 100% acetonitrile). The Q Exactive system was used for MS/MS analysis in the positive ion and information-dependent acquisition mode. Proteins were identified and quantified using Proteome Discoverer (v2.1, Thermo Fisher Scientific), using three embedded search nodes; Mascot^{72 (link)} (v2.5.1), Sequest HT (Thermo Fisher Scientific), and MS Amanda^{73 (link)} (v2.1.5) search algorithms. The Percolator algorithm was used to calculate the false discovery rate (FDR) of peptide spectrum matches, set to a q-value of 0.05^{74 (link)}. Entrez labels and gene names were retrieved with the R interface to Uniprot web services R package version 2.20.0. Raw counts were normalized using the weighted trimmed mean of M-values method^{75 (link)}, and the batch effect for the two iTRAQ experiments was removed using ComBat^{76 (link)}. The contrast between different conditions was carried out with the quasi-likelihood test implemented in edgeR^{77 (link)}. Raw data are accessible at 10.5281/zenodo.6140992.

Cell extracts were prepared using lysis buffer (1% NP-40, 150 mM NaCl, 100 mM Hepes, 5 mM Na₄P₂O₇, 5 mM NaF, 2 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride, 10 mg/l aprotinin, 10 mg/l leupeptin, PMSF).
For GST pull-down assay, the lysates were incubated with Glutathione-Sepharose 4B beads (GE Healthcare) at 4 °C 4 h on rotation. After three washes with lysis buffer, the bound proteins were released in SDS loading buffer.
For the Flag pull-down assay, the lysates were first incubated with anti-Flag M2 affinity gel (Sigma) at 4 °C overnight with rotation. After incubation, complexes were washed three times with lysis buffer, and the bead-conjugated proteins were released with SDS loading buffer.