Fig. 4

FBN1 combines directly with VEGFR2 in ovarian cancer cells. (A) Co‐IP assay of interactions between FBN1 and VEGFR2 proteins in cisplatin‐resistant ovarian cancer organoids and OVCA433‐CisR cells. (B‐C) Interactions between FBN1 and VEGFR2 confirmed by FRET‐FLIM upon transient co‐expression in OVCA433‐CisR cells. **, P < 0.01. (D) Western blotting assay for determining the relationship between FBN1 and VEGFR2, p‐VEGFR2, downstream molecules of VEGFR2‐mediated signaling in ovarian cancer cells. (E) Immunofluorescence assay for determining the relationship between FBN1 and p‐VEGFR2 (Tyr1054) in cisplatin‐resistant ovarian cancer organoids and cell lines. Green signals, FBN1; red signals, p‐VEGFR2 (Tyr1054); blue signals, DAPI. (F) Western blotting assay of p‐AKT1 (Ser473) in cisplatin‐resistant ovarian cancer organoids and OVCA433‐CisR cells. (G) Co‐IP assay demonstrated an interaction between VEGFR2 domains 2 & 3 and FBN1 protein. Binding of VEGFA and the extracellular domains 2 & 3) of VEGFR2 was used as the positive control. (H) Mutant codons and amino acids of D2 and D3 of VEGFR2. (I) Western blotting assay of p‐AKT1 (Ser473) in cisplatin‐resistant ovarian cancer organoids and OVCA433‐CisR cells. Abbreviations: FBN1, fibrillin‐1; CR, cisplatin‐resistant; KO, knockout; NC, negative control; SDS‐PAGE, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis; AKT, protein kinase B; Co‐IP, co‐Immunoprecipitation; VEGFR2, vascular endothelial growth factor receptor 2; DAPI, 2‐(4‐amidinophenyl)‐6‐indolecarbamidine dihydrochloride; FRET‐FLIM, Fӧrster resonance energy transfer‐fluorescence lifetime imaging. FE, FRET efficiency