L lysine

HEK293T cells were cultured for two weeks in SILAC DMEM lacking L-lysine and L-arginine and supplemented with 1% penicillin-streptomycin, 10% heat-inactivated dialyzed FBS, and 200 µg/mL L-proline (all purchased from Thermo Fisher Scientific). The medium was supplemented with 84 µg/mL L-arginine and 146 µg/mL L-lysine (both from Thermo Fisher Scientific) for the light medium (K0 R0), while the same amounts of ¹³C₆-Larginine and ²H₄-L-lysine, or ¹³C₆-¹⁵N₄-L-arginine and ¹³C₆-¹⁵N₂-L-lysine (from Cambridge Isotope Laboratories, Tewksbury, MA, USA, kindly provided by Guillaume Bossis, IGMM, Montpellier, France) were added to the medium and heavy media, respectively. After complete labeling monitored by MS analysis, 5 × 10⁶ cells from each condition were seeded in 10 cm Petri dishes. Light-labeled cells were transfected with an empty plasmid, whereas medium- and heavy-labeled cells were transfected with plasmids expressing Daxx wt or 1-732 (ΔSIM) mutant, respectively. Cells were transduced 24 h post transfection with VSV-G pseudotyped HIV-1 at a MOI of 10. After 4 h, cell extracts were prepared and Daxx was immunoprecipitated using anti-HA antibodies.

Polyethersulfone (PES) microfiltration membranes (0.45 μm pore diameter, 47 mm diameter) were purchased from MilliporeSigma (Burlington, USA). Glycidyl methacrylate (≥97.0%), lanthanum(III) nitrate hexahydrate (99.999% trace metal basis), cerium(III) chloride heptahydrate (99.9% trace metal basis), neodymium(III) nitrate hexahydrate (99.9% trace metal basis), zinc perchlorate hexahydrate, calcium chloride (anhydrous, Redi-Dri, ≥97%), sodium standard for ICP (1,000 mg/L ± 2 mg/L), calcium standard for ICP (1,000 mg/L ± 2 mg/L), and magnesium standard for ICP (1,000 mg/L ± 2 mg/L) were purchased from Sigma-Aldrich. Magnesium chloride hexahydrate (99–102%, ACS grade) was purchased from VWR Life Science. L-lysine (98%, powder) was purchased from Alfa Aesar. Ethyl alcohol (absolute, 200 proof, ≥99.5%, ACS Reagent grade) was purchased from Acros Organics. Denatured Ethyl alcohol (88–91%), sodium chloride (≥99%, certified ACS grade, crystalline), nitric acid (68–70%, certified ACS plus grade), and hydrochloric acid (36.5–38%, certified ACS plus grade) were purchased from Fisher Chemical. Arsenazo (III) was purchased from Pointe Scientific. Sodium hydroxide (10 N, biotech reagent grade) was purchased from Avantor. Deionized water (DI water) was made from a RiOS-DI 3 water purification system (MilliporeSigma, Burlington, MA, USA).

Indomethacin (IND, γ polymorph), l-histidine (HIS), l-lysine (LYS), l-isoleucine (ISO), glycine (GLY); 99% purity, were purchased from Alfa Aesar (Heysham, England). l-arginine (ARG); ≥99.5% purity, was purchased from Sigma Aldrich (Nottingham, United Kingdom). All powders were used as received from the supplier, and properties of the raw chemicals are described in Table 1. Ultrapure water was produced from a Pure Lab Ultra-System (London, United Kingdom), hydrochloric acid 32% and sodium hydroxide; ≥97% purity, were supplied by Sigma Aldrich (Poole, United Kingdom) and VWR international (Leicestershire, United Kingdom), respectively. A pH meter (OXOID limited, England) was calibrated with standard buffer solutions: phosphate buffers; pH 7 and 4 ± 0.2 at 25 ± 1 °C and used to determine the pH of the solutions. A temperature probe (YC-747UD data logger thermometer) was used to accurately determine the solution temperature (precision 0.1 °C).