P1860

The protocol for co-immunoprecipitation was previously described ^{3 (link)}. Briefly, a confluent plate of HEK293 cells was split 1:1.5 into 6-well plates and transfected with a DNA-Lipofectamine 2000 mixture (0.5–4 μg of DNA and 5 μl of Lipofectamine 2000 in 250 ml of DMEM per well) 22–24 hr before cell lysis. Transfection efficiency was monitored based on GFP emissions. HEK293 cells transiently expressing GFP, GFP-tagged pUL36 mutants, and HA-tagged kinesin light-chain isoforms, were treated with 10 μM nocodazole (VWR) for 1 hr and lysed in 500 μl/well of cold TAP lysis buffer (50 mM Tris-HCl pH 7.5, 50 mM KCl, 2 mM MgCl₂, 0.1 mM EDTA, 5% glycerol, 1% NP-40) supplemented with 10 μM nocodazole, 1 mM dithiothreitol, 0.5 mM NaF, 0.1 mM Na₃VO₄, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and a protease inhibitor cocktail [P1860, Sigma]. Cell lysates were centrifuged at 14,000 rpm for 20 min at 4°C. GFP-tagged proteins were immunoprecipitated using an anti-GFP [Invitrogen, A-6455] antibody-loaded Sepharose A/G bead slurry [CalBiochem, IP05] overnight (16–18 h) at 4°C. Beads were washed four times with cold supplemented TAP lysis buffer and resuspended in 60 μl of 2x final sample buffer supplemented with 10% β-mercaptoethanol. Samples were either immediately used for western blot analysis, or heated at 85°C for 15 min and then stored at −20°C.

PMVECs were grown to approximately 70% confluence in a 6-well plate (3 mL per well). Isolated I/R EVs (2.6 × 10⁹ EVs per well) were resuspended in EV-free media, added to two wells and incubated for 10 min at 37 °C. Two wells were treated with 10 μM rolipram and incubated at 37 °C for 5 min, then treated with 1 μM isoproterenol and incubated at 37 °C for 10 min. Two wells were treated with vehicle and incubated at 37 °C for 5 min, then treated with serum free media and incubated at 37 °C for 10 min. Cells from each of the 6 wells were lysed on ice in RIPA buffer containing 1% protease inhibitor (Sigma, P1860) and 1% phosphatase inhibitor (Sigma P0044). Cells were scraped with a cell scraper and aspirated into a syringe with a 25G needle, then transferred to microfuge tubes. Cell lysates were centrifuged at 10,000 ×g for 10 min at 4 °C and pellet was discarded.
PMVECs were pretreated with 25 μM propranolol (a non-selective β-blocker) for 1 h to inhibit β-receptors. Following pretreatment, PMVECs were treated with either isoproterenol or I/R stimulated EVs as above for 10 min. Whole cell lysates were collected and analyzed for VASP/p-VASP and PKA activity as described below.