![Figure 5. ANXA2 associates with VEC, VE-PTP, and SHP2 and supports phosphatase function. (A) Confocal image of lung section stained with FITC anti–ANXA2 IgG (green) and Cy3 anti–VEC IgG (red, left; enlarged, right). Subpanels outlined in red show no primary control–stained section for anti-ANXA2 (*) and anti-VEC (**). Bar, 10 µm. (B) IEM image of pulmonary microvessel stained with anti-ANXA2 and anti-VEC (10-nm and 6-nm gold conjugates, blue and red arrows, respectively, left; enlarged, right). Bars: (left) 100 nm; (right) 50 nm. (C) Lung tissue extracts immunoprecipitated (IP) with anti–VEC or nonimmune IgG and immunoblotted for VEC, ANXA2, and ANXA4. (D) Confocal image of lung section stained with stained with FITC anti–ANXA2 IgG (green) and Cy3 anti–VE-PTP IgG (red, left; enlarged, right). Subpanels outlined in red show no primary control–stained section for anti-ANXA2 (*) and anti-VE-PTP (**). Bar, 10 µm. (E) IEM images of pulmonary microvessel stained with anti-PTP and anti-ANXA2 (10-nm and 6-nm gold conjugates, red and blue arrows, respectively, left; enlarged, right). Bars: (left) 500 nm; (right) 200 nm. (F) Pulmonary microvessel stained with anti-PTP and anti-VEC followed by gold-conjugated (10- and 6-nm conjugates, red and blue arrows, respectively, left; enlarged, right). Bars: (left) 200 nm; (right) are 50 nm. (G) Miles assay (conducted as described in Materials and methods) to assess VEGF-induced cutaneous EB extravasation in Anxa2+/+ and Anxa2−/− mice (n = 7–8 mice per group, ANOVA). (H) Effect of Na3VO4 (sodium orthovanadate [OV]) or 100 µl, 5 mM sodium fluoride (NaF; subcutaneously, n = 8 mice per group, ANOVA) on VEGF-induced cutaneous EB extravasation. (I) Extracts of lung tissue immunoprecipitated with anti-VEC and immunoblotted for VEC, VE-PTP, SHP2, and ANXA2. (J) Quantification of immunoprecipitated bands in I (data combined from four experiments, Student’s t test). (K) Extracts of lung tissue were immunoprecipitated with anti–VE-PTP and immunoblotted for VE-PTP, VEC, ANXA2, and SHP2. (L) Quantification of immunoprecipitated bands in K (data combined from four experiments, Student’s t test). Data in A–I and in J–L are representative of three and four experiments, respectively. Black and white bars indicate Anxa2+/+ and Anxa2−/− mice, respectively. Data are expressed as mean ± SEM. *, P < 0.05; ***, P < 0.001.](https://cdn.rupress.org/rup/content_public/journal/jem/214/9/10.1084_jem.20160652/8/jem_20160652_fig5.jpeg?Expires=1767643199&Signature=d99Y3Gs~0i8kLIzHvd79I5eE~~t-lfOFfWns3yBc0gZdURLclumFPAaxjhC5~XKoZ5u6KySQgVK10x9MUYmbD~8ZZAy-ud5xmoe0z3tTfk9vN4it9XLw6RnnbmmXPDADWxVRLpnowai3-APwDbDOgTZ6Sw1Y1T6hPkz~ccUy4F5a-7BPzoMnZME3VlXW4Nv~s8uLhtx3wcR-u02ziUmsCsz8zxUQGpDrwyjif6n14quEpzBh11tMhb7iq4M9ZznyVIje~lVSmsiZFxJN6DUBD0NrRdxohaz-Mi9~pwOM67lJDH0mnPWOLgoQIkfNcRKbMvo3758r9c19rJvBX7~XTA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
ANXA2 associates with VEC, VE-PTP, and SHP2 and supports phosphatase function. (A) Confocal image of lung section stained with FITC anti–ANXA2 IgG (green) and Cy3 anti–VEC IgG (red, left; enlarged, right). Subpanels outlined in red show no primary control–stained section for anti-ANXA2 (*) and anti-VEC (**). Bar, 10 µm. (B) IEM image of pulmonary microvessel stained with anti-ANXA2 and anti-VEC (10-nm and 6-nm gold conjugates, blue and red arrows, respectively, left; enlarged, right). Bars: (left) 100 nm; (right) 50 nm. (C) Lung tissue extracts immunoprecipitated (IP) with anti–VEC or nonimmune IgG and immunoblotted for VEC, ANXA2, and ANXA4. (D) Confocal image of lung section stained with stained with FITC anti–ANXA2 IgG (green) and Cy3 anti–VE-PTP IgG (red, left; enlarged, right). Subpanels outlined in red show no primary control–stained section for anti-ANXA2 (*) and anti-VE-PTP (**). Bar, 10 µm. (E) IEM images of pulmonary microvessel stained with anti-PTP and anti-ANXA2 (10-nm and 6-nm gold conjugates, red and blue arrows, respectively, left; enlarged, right). Bars: (left) 500 nm; (right) 200 nm. (F) Pulmonary microvessel stained with anti-PTP and anti-VEC followed by gold-conjugated (10- and 6-nm conjugates, red and blue arrows, respectively, left; enlarged, right). Bars: (left) 200 nm; (right) are 50 nm. (G) Miles assay (conducted as described in Materials and methods) to assess VEGF-induced cutaneous EB extravasation in Anxa2+/+ and Anxa2−/− mice (n = 7–8 mice per group, ANOVA). (H) Effect of Na3VO4 (sodium orthovanadate [OV]) or 100 µl, 5 mM sodium fluoride (NaF; subcutaneously, n = 8 mice per group, ANOVA) on VEGF-induced cutaneous EB extravasation. (I) Extracts of lung tissue immunoprecipitated with anti-VEC and immunoblotted for VEC, VE-PTP, SHP2, and ANXA2. (J) Quantification of immunoprecipitated bands in I (data combined from four experiments, Student’s t test). (K) Extracts of lung tissue were immunoprecipitated with anti–VE-PTP and immunoblotted for VE-PTP, VEC, ANXA2, and SHP2. (L) Quantification of immunoprecipitated bands in K (data combined from four experiments, Student’s t test). Data in A–I and in J–L are representative of three and four experiments, respectively. Black and white bars indicate Anxa2+/+ and Anxa2−/− mice, respectively. Data are expressed as mean ± SEM. *, P < 0.05; ***, P < 0.001.
