L 2 3 3h proline

nrCFBs were seeded in 12-well plates and transduced with Veh or L3 after 24 h. The medium was changed after 24 h to serum-free medium containing 50 µM/mL ascorbic acid and 1 µCi L-[2,3-³H]-Proline (Cat# NET323001 MC, PerkinElmer, Inc, MA). On day 4, cells were washed with cold PBS, lysed in 1M NaOH and diluted using the OptiPhase HiSafe 3 liquid scintillation cocktail (Cat# 1200.437, PerkinElmer). The amount of radiolabelled proline present in each sample, as a surrogate for collagen protein biosynthesis^{45 (link)}, was measured as counts per minutes (CPM) using the Wallac Winspectral 1414 liquid scintillation counter (PerkinElmer).

Proline incorporation assays were performed as described [18 (link)]. Fibroblasts were seeded at a density of 10’000 cells/well onto gelatin-coated 96-well plates in 100 μl/well complete growth medium. The next day the medium was exchanged for 100 μl/well starvation medium. After 24h incubation, cells were treated and supplemented with 10 μl/well of L-[2,3-³H]-proline in starvation medium (0.2 μCi/well, Perkin Elmer) at the same time and cultivated for 24h. For determination of [³H]-proline incorporation, cell supernatants were discarded, cells were lysed in 150 μl/well NaOH (0.15 M) and lysates were incubated on ice for 30 min. For protein precipitation, 100 μl/well of trichloroacetic acid (50% (w/v) stock, final concentration 20%) was added and the lysates were incubated on ice for 30 min. Precipitated proteins were collected with the Filtermate cell harvester (Perkin Elmer) onto glass fiber filters (Unifilter-96, GF/C) (Perkin Elmer), filters were washed eight times with deionized water, dried and then supplemented with 60 μl/well of liquid scintillator Microscint20 (Perkin Elmer). Plates were subjected to liquid scintillation counting using TopCount (Perkin Elmer).

For the cell‐free synthesis of radioligands one amino acid was replaced with a tritiated variant depending on its abundance in the respective ligand. Complement 5a was produced in the presence of 1 μm (FM)/2.1 μm (RM) L‐[2,3‐³H]‐proline (specific activity 55 Ci·mmol⁻¹, Perkin Elmer, Boston, USA). For labelling of endothelin‐1 1 μm (FM)/0.9 μm (RM) L‐[2,3,4,5,6‐³H]‐phenylalanine (specific activity 128.1 Ci·mmol⁻¹, Perkin Elmer) was added. The cell‐free reaction was initiated omitting the labelled amino acid and incubated at 30 °C, 100 rpm for 3 h. The reaction mix was removed from the D‐tube^™ dialyzer and incubated with 0.2 mL Ni‐NTA magnetic agarose beads suspension (Qiagen) per mL reaction mix for 10 min to remove undesired side products translated with endogenously present amino acids from the S30 extract. The reaction mix was removed from the magnetic beads, supplemented with 0.25 mm L‐histidine to account for potential losses in the IMAC (immobilized metal ion affinity chromatography) step and the tritium‐labelled amino acid. The feeding mix was discarded and replaced with a fresh mix containing the labelled amino acid. Reactions were incubated at 30 °C in a water bath under constant shaking overnight.