Keratinocytes were lysed in radioimmunoprecipitation assay (RIPA) buffer for 4 hours on ice and then centrifuged for 10 min at 14.000 rpm at 4°C. Forty micrograms of cell lysate was loaded in 4/12% bis/tris-acrylamide gel (Invitrogen) and separated by electrophoresis. Proteins were transferred on polyvinylidene difluoride (PVDF) membranes. The membranes were incubated overnight with anti-Nrp1 (goat, 1:200, R&D, catalog number AF 566) or anti-Flt1 (mouse, 1:1000, Santa Cruz, clone H-225, catalog number sc-9029) or anti-Fosl1 (mouse, 1:1000, Santa Cruz, clone C12, catalog number sc-28310) or Flt1 (rabbit, 1:1000, Abcam, ab3252) or anti–phospho-Flt1 Y1213 (rabbit, 1:1000, R&D, catalog number AF4170), (mouse, 1:1.000, Cell Signaling, catalog number 9106), anti–HA (human influenza hemagglutinin tag) (mouse, 1:1000, Sigma-Aldrich, catalog number: 11583816001), and anti–β-actin (1:3000, Abcam, catalog number ab8227). Anti-mouse or anti-rabbit immunoglobulin G (IgG) conjugated with horseradish peroxidase (HRP) (1:3000 or 1:10,000; Healthcare) was used as the secondary antibody.

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