ERK and Akt pathway activation analysis
LYSED, SECONDARY
To verify whether LEAP‐purified pEVs treatment induced ERK or Akt phosphorylation, AlphaLISA assays were performed according to the manufacturer's recommendations to quantify the amount of total and phosphorylated ERK and Akt proteins present in dermal fibroblasts after treatment with pEVs. Briefly, normal human dermal fibroblasts were either untreated or treated with pEVs (15 μg/mL). After 1, 2.5, 5, 15, 30, and 60 min of treatment, cells were lysed, and resulting lysates were used as analytes in the AlphaLISA assays run in 384‐well plates (OptiPlate, PerkinElmer). Acceptor beads conjugated with biotinylated antibodies were added to the samples and allowed to incubate in the dark for 60 min at room temperature. Streptavidin‐coated donor beads were added, followed by a secondary incubation in the dark for 60 min. The AlphaLISA signal was read on the Clariostar spectrophotometer at an excitation wavelength of 680 nm.