Ficoll 70

In 7 NGF experiments, the nAChR antagonist mecamylamine (mecamylamine hydrochloride; Sigma, USA), which is blood–brain barrier (BBB) permeable, was used. mecamylamine was dissolved in saline at a concentration of 20 mg/ml, and administered intravenously through a femoral vein as a single dose at 100 min before the onset of the brushing stimulation. Ficoll 70 (Pharmacia Biotech, Sweden) was injected (i.v.) to compensate hypotension following mecamylamine injection. In preliminary experiments, we confirmed that mecamylamine did not produce systematic changes in NGF levels in cortical perfusate throughout 600 min after injection.

Polar extract of E. coli lipids and l-α-phosphatidylcholine from Avanti Polar Lipids (Alabaster, AL, USA) and Sigma, respectively, were dissolved in spectroscopy-grade chloroform and stored at −20°C. Silica microbeads (∼5 μm mean diameter) were purchased from Bangs Laboratories, Inc. (Fishers, IN). Ficoll 70 from GE Healthcare was equilibrated by dialysis in 50 mM Tris-HCl (pH 7.5), 100 mM KCl, and the final concentration was calculated from its refractive index increment (29 (link)). Alexa Fluor 488 and Alexa Fluor 647 carboxylic acid succinimidyl ester dyes were from Molecular Probes/Thermo Fisher Scientific. Analytical-grade chemicals were from Merck.

Dextran T40 was from Amersham Pharmacia Biotech, Ficoll 70 was from General Electric, and PEG 20 and sucrose were from Merck. PEG 8, ribonuclease A (RNase A, 9.3 isoelectric point [20 (link)]) and salmon testes DNA were from Sigma. Ovomucoid (4.3 isoelectric point [21 ]) was from Worthington Chemicals. All crowders were used without further purification, equilibrated by extensive dialysis in the working buffer (50 mM Tris-HCl, pH 7.5, 500 mM KCl, 100 μM MgCl₂). Final concentrations of the stock solutions were determined from the refractive index increment (Dextran T40, 0.147 mL/g (APS Corp.); Ficoll 70, 0.141 mL/g [22 (link)]; PEG 20 and PEG 8, 0.136 mL/g [23 (link),24 (link)]; and sucrose, 0.138 mL/g [25 ]) or spectrophotometrically (RNase A, ε₂₈₀ = 9440 M^-1 cm^-1 (calculated from the sequence); ovomucoid, ε₂₈₀ = 11275 M^-1 cm^-1 [26 (link)]). The water of the commercial 10 g/L DNA solution was evaporated using a SpeedVac and the pellet dissolved in the desired volume of working buffer to reach the final concentration.

The method for the evaluation of the aggregation propensity followed those used in previous comprehensive analysis (Niwa et al., 2009 (link)). The template DNA for expression by the cell-free translation system was amplified from an E. coli ORF library [ASKA library (Kitagawa et al., 2005 (link); Riley et al., 2006 (link))] by PCR, as described previously (Niwa et al., 2009 (link)). The transcription-translation-coupled expression was conducted by a reconstituted cell-free translation system [PURE system (Shimizu et al., 2001 (link), 2005 (link))] at 37°C for 1 h. For detection, L-[³⁵S]-methionine was added to the PURE system. Ficoll 70 (GE Healthcare) or dextran 70 (Sigma–Aldrich) was also included at the concentration of 80 mg/ml in the reaction, to evaluate the effects of MCRs. After the expression, an aliquot was withdrawn as the total fraction, and the remainder was centrifuged at 20,000 × g for 30 min. The total and supernatant fractions were separated by SDS-PAGE, and the band intensities were quantified by autoradiography (FLA7000 image analyzer and Multi Gauge software, Fujifilm). The ratio of the supernatant to the total protein was defined as the solubility, as referred to as the index of aggregation propensity.

Lipid and content mixing assays, using v- and t-SNARE liposomes, were carried out as previously described^{72 (link)} except that the macromolecular crowding agent^{49 (link)}, Ficoll 70 (100 mg/ml, GE Healthcare), was included in the reconstitution buffer (25 mM HEPES, 100 mM KCl, pH 7.4). Data were obtained from three independent trials.

Ampicillin and isopropyl-β-D-1-tiogalactopyranoside were obtained from Apollo Scientific (Stockport, United Kingdom). Imidazole, bovine serum albumin (BSA), Trizma base, TSP ((trimethylsilyl)-2,2,3,3-tetradeuteropropionic acid), SIGMA-FAST-EDTA-free Protease inhibitor, 2,2,2-trifluorethanol (TFE), the ¹⁵NH₄Cl salt and His-Select HF nickel resin were from Sigma-Aldrich (Madrid, Spain). Triton X-100, the paramagnetic probe S-(1-oxyl.2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl) methyl methane sulfonothioate (MTSL), ultra-pure urea, ultra-pure guanidinium hydrochloride (GdmCl), polytetrafluoroethylene (PTFE) filters with a size of 0.22 μm, and protein marker (PAGEmark Tricolor) were from VWR (Barcelona, Spain). Dextran-40, Ficoll-70 and PD-10 desalting-columns were acquired from GE Healthcare (Barcelona, Spain). Bio-Rad (Madrid, Spain) column sleeves were used for the Ni-immobility affinity column chromatography step. Amicon centrifugal devices with a cut-off molecular weight of 3 kDa were from Millipore (Barcelona, Spain). The rest of the materials were of analytical grade. Water was deionized and purified on a Millipore system.