Sonorex rk 156

Before harvesting, the AV leaflets were starved for 4 h in cultivation medium without FCS. Afterwards the leaflets of each replicate were bisected axially and perpendicularly to the free edge, whereas one half was stimulated with 100 nM insulin (acute insulin, AI) in PBS (Thermo Fisher Scientific) for 10 min to activate insulin-signaling pathways, while the other one remained in PBS without insulin as untreated condition. Appropriate conditions were evaluated in preliminary experiments (Supplemental Figure S4: Duration of acute insulin stimulation). After stimulation AV were snap-frozen, mechanically homogenized and solved in RIPA lysis buffer including PhosStop (Sigma Aldrich) and protease inhibitors (cOmplete, Sigma Aldrich). Cell disruption was carried out by sonification (Sonorex RK 156, Bandelin electronic, Berlin, Germany) for 15 min followed by centrifugation in order to remove the cell debris. The total protein concentration was determined by DC assay (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions.

Freeze-dried filters containing cyanobacterial material and the cell-bound fraction of MCs were extracted with 2.4 mL of 75% methanol for MCs (Spoof et al. 2003 (link), Hautala et al. 2013 (link)). The extracts were sonicated for 15 min in a bath sonicator (Bandelin Sonorex RK 156, Berlin, Germany) and additionally for 1 min with a probe sonicator (Bandelin Sonopuls HD 2070 with a 3-mm microtip, 30% pulse, 30% energy). After centrifugation at 10,000g for 10 min, part of the supernatant was concentrated by evaporation with nitrogen gas at 50 °C. The residue was resuspended in 75% methanol (150 μL) and clarified by filtration through an Acrodisc GHP 0.45 μm syringe filter (Pall Life Sciences, Ann Arbor, MI, USA) prior to MC analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
The OASIS HLB SPE cartridges prepared at the site of sample collection and intended to capture to the extracellular MCs were eluted with 3 mL of 100% methanol. The methanol was evaporated with nitrogen gas at 50 °C. The dry residue was resuspended in 75% methanol (150 μL) and clarified by filtration through an Acrodisc GHP 0.45-μm syringe filter prior to LC-MS/MS.