Amicon ultra 2

Viral RNA was extracted from 140 μL plasma or serum using QIAamp viral RNA extraction mini-kits (Qiagen Ltd) and eluted with 60 μL RNase-free water. Urine and saliva were concentrated (Amicon Ultra-2, Millipore) before RNA extraction. Samples were eluted in 30 μL RNase-free water/mL of urine, stored at −80 °C until assayed. 40–50 mg tissue was disrupted and homogenised using a bead mill Tissue Lyser (Qiagen Ltd). Total RNA was extracted using an RNA Plus Universal mini kit (Qiagen Ltd). Tissue RNA quantified by nanodrop spectrophotometry was stored at −80 °C.

AtT20 cells stably expressing wild-type or mutant provasopressin or A1Pi were grown in 10-cm dishes, washed with PBS, and then incubated for 30 min in 3 ml secretion medium (Earle’s balanced salt solution without Ca²⁺ and Mg²⁺, supplemented with MEM amino acids, 2 mM l-glutamine, MgSO₄, and 2 mM CaCl₂; Sigma). After collecting the media, the cells were washed with PBS and incubated for another 30 min in stimulation medium (secretion medium without CaCl₂, but containing 2 mM BaCl₂). Collected secretion and stimulation media were centrifuged for 15 min at 3000 × g, and the supernatants were concentrated using Amicon Ultra-2 (molecular weight cut-off 3 kDa; Millipore). Samples were analyzed by immunoblotting.

Conjugation was performed as previously described [17 ]. Shortly, m11B6 (provided by the University of Turku, Finland) in 0.07 M sodium borate buffer (Sigma Aldrich, St Louis, MO, USA), pH 9.2, was concentrated on an Amicon Ultra-2 centrifugal filter, 2 mL, 100 K, (Millipore, Billerica, MA, USA) and conjugated at 40 °C with the chelator CHX-A''-DTPA (Macrocyclics, Dallas, TX, USA) in a chelator to antibody molar ratio of 3:1 for 4 h. The reaction was terminated and CHX-A''-DTPA-m11B6 was separated from free chelate by size-exclusion chromatography using a NAP-5 column (GE Healthcare, Uppsala, Sweden), equilibrated with 20 mL of 0.2 M ammonium acetate buffer pH 5.5. The conjugated antibody was kept at −20 °C for labeling.

Approximately 0.5 ml of 5 mg ml^–1 α-agarase AgaWS5 was incubated with 1.5 ml of 1.5% agarose at 40°C in 1 mM potassium phosphate buffer (pH 7.0) for 12 h. The reaction mixture was cooled at 4°C for 10 min, was centrifuged at 15,000 × g for 30 min, and then the supernatant was filtered with a Millipore Amicon ultra-2 centrifugal filter unit for protein removal. The filtered liquid containing agarotetraose was collected and stored in −20°C. The agarotetraose was then identified by TLC and mass analysis.

The NCP (0.1 μM) and p300(BRPH_ΔAILZ) (2 μM) were incubated at 25°C for 30 min in buffer [20 mM HEPES-NaOH (pH 7.5), 20 mM NaCl, 0.5 mM MgCl₂, 1 μM Zn(OAc)₂, 1 mM dithiothreitol, 0.03% NP-40, and 0.5% glycerol]. The sample was then crosslinked by adding 2.5% glutaraldehyde to a final concentration of 0.1%, and incubated at 4°C for 30 min. The crosslinking reaction was quenched by adding 1 M Tris-HCl (pH 7.5) to a final concentration of 50 mM and incubating it on ice for 10 min. The crosslinked sample was applied onto the top of a sucrose density gradient [5–20% sucrose gradient in 10 mM Tris-HCl (pH 7.5), 30 mM NaCl, 1 μM Zn(OAc)₂, and 1 mM dithiothreitol] and centrifugated at 27,000 r.p.m., at 4°C for 16 h in an SW 41 Ti rotor (Beckman Coulter). After the ultracentrifugation, aliquots (630 μL) were collected from the top of the gradient and analyzed by 4% non-denaturing polyacrylamide gel electrophoresis in 0.5×TBE buffer, followed by SYBR Gold staining. The fractions containing the p300(BRPH_ΔAILZ)-NCP complexes were combined, and then desalted using a PD-10 column (Cytiva) in final buffer [10 mM Tris-HCl (pH 7.5), 30 mM NaCl, and 1 mM dithiothreitol]. The sample was then concentrated with an Amicon Ultra-2 centrifugal filter unit (Merck, 30,000 MWCO) and stored on ice.