Fig. 5

PTN97–110 inhibits VEGFA165-induced signaling in endothelial cells. (A) HUVEC were treated with VEGFA165 (10 ng/ml) and/or PTN97-110 (100 ng/ml) for 10 min and cell lysates were analyzed by antibodies against Akt phosphorylated at Ser473 (pAkt), and total Akt (tAkt). A representative Western blot is shown (n = 3). Results are expressed as the percent pAkt/tAkt ratio compared to untreated HUVEC (set as default = 100). (B) HUVEC were treated with VEGFA165 and/or PTN97-110 for 10 min and cell lysates were analyzed by antibodies against VEGF receptor 2 phosphorylated at Tyr1175 (pVEGFR2), and total VEGF receptor 2 (VEGFR2). A representative Western blot is shown (n = 3). Results are expressed as the percent pVEGFR2/VEGFR2 ratio compared to untreated HUVEC (set as default = 100). (C) Formation of tyrosine phosphorylated VEGF receptor 2 (pVEGFR2) complexes as evidenced by in situ PLA in HUVEC cultured in the presence or absence of VEGFA165 and/or PTN97-110. Representative pictures are shown. The scale bar corresponds to 10 μm. The box plots indicate the detected signals' median, mean, and range (n = 8–10 image fields with 4–8 cells per image per sample type, each sample run in triplicate). (D) HUVEC were treated with VEGFA165 and/or PTN97-110 for 10 min and cell lysates were analyzed by antibodies against ERK1/2 phosphorylated at Tyr895 (pERK1/2), and total ERK1/2 (tERK1/2). Representative Western blots are shown (n = 3). Results are expressed as mean ± SD of the percent pERK1/2/tERK1/2 ratio compared to untreated HUVEC (set as default = 100).