hFlt1-HEK293 lysates were immunoprecipitated by incubation with anti-HA (1 μg of antibody per 1.5 mg of protein, rabbit,, Abcam, ab9110) coupled to Protein G Dynabeads (Invitrogen, catalog number 10003D) overnight at 4°C. Proteins were eluted in sample buffer by heating to 70°C for 10 min. For Western blot analysis, equal amounts of total protein were loaded and resolved on a NuPAGE 4/12% bis/tris gel (Invitrogen) and transferred to a PVDF membrane. Blots were probed with specific antibodies for HA-tag (mouse, 1:1000, Sigma, catalog number 11583816001), Flt1 (rabbit, 1:1000, Abcam, ab3252), and anti-PFlt1 (rabbit, 1:1.000, R&D, Y1213). Enhanced chemiluminesence anti-mouse or anti-rabbit IgG conjugated with HRP (1:3000 or 1:10,000; Healthcare) was used as the secondary antibody.

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