L arginine hcl

Aortic valves were preserved and transported in PBS, and processed within a maximum of 2 h after extraction. The valves were washed immediately in PBS to reduce blood contaminants. For 2D-DIGE and western blotting, one aortic valve leaflet was ground into a powder in liquid N₂ in a mortar. Protein extracts were then prepared from the valve as described previously [8 (link), 9 (link)] and the total protein concentration was measured by the Bradford-Lowry method (Bio-Rad protein assay) [10 ].
For secretome analysis, the second valve leaaflet was cultured as described elsewhere [11 (link), 12 (link)] using medium supplemented with antibiotics and amphotericin B (Fungizone^®) to avoid contamination. Samples were transferred to a Petri dish (Cell Star^®), cut into pieces and incubated at 37 °C in lysine-arginine free 1640 RPMI medium (Cell Culture Technologies Invitrus) supplemented with 5 mg/ml fungizone, 250 mg/ml amikacin, 2 mg/ml l-lysine 2HCl (U-13C6, 97–99%) and 10 mg/ml l-Arginine HCl (U-13C6, 97–98%: Cambridge Isotope Laboratories Inc., Andover, MA) in a humidified atmosphere of 5% CO₂. The valves were cultured for 96 h and the medium collected was stored at −80 °C until analysis. Finally, the valves were fixed in formalin at 4 °C, decalcified in Shandon-TBD1 (Thermo Scientific) and embedded in OCT for subsequent immunohistochemistry.

SILAC RPMI 1640 media (Thermo Scientific Pierce) was supplemented with 240 mg/mL of L-arginine‐HCl (¹³C₆¹⁵N₄) and 40 mg/mL of L‐lysine‐2HCl (¹³C₆¹⁵N₂) (Cambridge Isotope Labs), 10% dialysed FBS (Life Technologies) and 1% Penicillin-Streptomycin (Gibco). The medium was sterile filtered through a 0.22 micron filter. TPC1 thyroid cancer cells were cultured for at least eight doublings at 37°C in a humidified atmosphere with 5% CO₂. Upon reaching 70-90% confluence, cells were washed with phosphate buffered saline (PBS), harvested and stored at -80°C until use. Heavy amino acid incorporation was verified by MS.

The isotopic labeling procedure was similar to that previously described by Zhang et al.^{35 (link)} The media utilized for isotopic labeling was Eagle’s Minimum Essential Medium (ATCC) supplemented with 15% dialyzed FBS (Thermo Scientific), 100 U/mL penicillin, and 100 U/mL streptomycin. Cells were gradually adapted to this media by replacing normal FBS with dialyzed FBS within five passages. Cells were then plated at a density of 1,000,000 cells per 10-cm plate.
One day after plating, the dividing cultures were switched to MEM labeling media for SILAC (Thermo Scientific) supplemented with l-arginine:HCl (¹³C6, 99%) and l-lysine:2HCl (¹³C6, 99%; Cambridge Isotope Laboratories) at concentrations of 0.1264 g/L and 0.087 g/L, 15% dialyzed FBS (Thermo Scientific), 100 U/mL penicillin, and 100 U/mL streptomycin. For whole proteome analysis, cells were collected after 0, 1, 2, 3, and 5 days of labeling and washed with PBS. For analysis of isolated PrP^Sc aggregates, cells were collected after 0, 1, 2, 3, and 4 days of labeling and washed with PBS. All cell pellets were frozen before further analysis.

For SILAC experiments, cells were cultured as described above with the following modifications. Culture media consisted of DMEM for SILAC (Fisher Scientific, Loughborough, UK) supplemented with L-arginine:HCL (¹³C₆, 99%; ¹⁵N₄, 99%) and L-lysine:2HCl (¹³C₆, 99%) (Cambridge Isotope Laboratories Inc., Tewksbury, MA, USA) and 10% dialysed FBS (Fisher Scientific), 1% penicillin/streptomycin and 1% L-glutamine. Control cells were grown in the labelled media and incubated with PBS for 8 h. Cells treated with Kdo2-lipid A and ATP were grown in the unlabelled media as described above. All cells were collected and pelleted and stored at -20 °C until processed. Cells were lysed and protein extracts from labelled and unlabelled cells mixed in stoichiometric amounts.