Fig. 6

Antibodies against the PTPRZ1 FNIII domain inhibit PTN and VEGFA165-induced endothelial cell migration and signaling. (A) HUVEC were incubated in the presence or absence of anti-FNIII (10 ng/ml) or mouse IgG (10 ng/ml) for 24 h. Total cell lysates were then immunoprecipitated by using an antibody against the C-terminus of PTPRZ1 or non-specific IgG and analyzed by Western blot using an antibody against PTN or VEGFA. The content of β-actin in the corresponding lysate was used as a loading control. Representative images from two independent experiments are shown. (B) Migration of HUVEC in the presence or absence of PTN (100 ng/ml), VEGFA165 (10 ng/ml), and/or anti-FNIII (10 ng/ml). Results are expressed as mean ± SD (n = 4) of the percent number of migrated cells compared to the untreated cells (set as default 100%). (C) Migration of HUVEC in the presence or absence of PTN, VEGFA165, and/or anti-PTPRZ1 (10 ng/ml). Results are expressed as mean ± SD (n = 5 for VEGFA165 and 3 for PTN) of the percent number of migrated cells compared to the untreated cells (set as default 100%). (D) HUVEC were treated with VEGFA165 and/or anti-PTPRZ1 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. Results are expressed as mean ± SD (n = 2) of the percent pAkt/tAkt ratio compared to untreated HUVEC (set as default = 100). (E) HUVEC were treated with VEGFA165 and/or anti-PTPRZ1 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. Results are expressed as mean ± SD (n = 3) of the percent pVEGFR2/VEGFR2 ratio compared to untreated HUVEC (set as default = 100).