Figure 4. AMIGO2 regulates subcellular... | Rockefeller University Press

Figure 4.

$Figure 4. AMIGO2 regulates subcellular localization and interacts with PDK1. (A) PDK1 immunostaining was performed in AMIGO2-inhibited HUVECs in the presence or absence of VEGF. Cells were starved for 6 h and treated with VEGF. White arrowheads indicate membrane localization of PDK1. Bars, 20 µm. (B) Subcellular localization analysis was performed by cell fractionation in VEGF-treated, AMIGO2-deficient HUVECs. (C) Quantification of Western blots. Subcellular localization analysis was performed by cell fractionation with or without VEGF in control siRNA and AMIGO2-deficient HUVECs. ***, P < 0.0001. Data are means ± SD. (D) Endogenous AMIGO2 interacts with PDK1 in HUVECs. AMIGO2 was immunoprecipitated and blotted with anti-PDK1 and -Akt antibodies. (E) Colocalization of PDK1 and AMIGO2 in the plasma membrane and cytosol of HUVECs in the presence of VEGF. HUVECs were starved for 6 h and stimulated with or without VEGF. White arrowheads indicate membrane edge areas of HUVECs. Bars, 20 µm. (F) HUVECs were starved for 6 h, stimulated with VEGF for 4 h, and immunoprecipitated with an AMIGO2 antibody and blotted with endogenous PDK1. (G) AMIGO2-deficient HUVECs were immunoprecipitated with an AMIGO2 antibody and blotted with an anti-PDK1 antibody. (H) Flag-PDK1 was immunoprecipitated and blotted with an anti-GFP antibody tagged to AMIGO2. A2, AMIGO2; HC, heavy chain; IB, immunoblotting; IgG, normal IgG; IP, immunoprecipitation; ns, not significant; WCL, whole cell lysate.$

AMIGO2 regulates subcellular localization and interacts with PDK1. (A) PDK1 immunostaining was performed in AMIGO2-inhibited HUVECs in the presence or absence of VEGF. Cells were starved for 6 h and treated with VEGF. White arrowheads indicate membrane localization of PDK1. Bars, 20 µm. (B) Subcellular localization analysis was performed by cell fractionation in VEGF-treated, AMIGO2-deficient HUVECs. (C) Quantification of Western blots. Subcellular localization analysis was performed by cell fractionation with or without VEGF in control siRNA and AMIGO2-deficient HUVECs. ***, P < 0.0001. Data are means ± SD. (D) Endogenous AMIGO2 interacts with PDK1 in HUVECs. AMIGO2 was immunoprecipitated and blotted with anti-PDK1 and -Akt antibodies. (E) Colocalization of PDK1 and AMIGO2 in the plasma membrane and cytosol of HUVECs in the presence of VEGF. HUVECs were starved for 6 h and stimulated with or without VEGF. White arrowheads indicate membrane edge areas of HUVECs. Bars, 20 µm. (F) HUVECs were starved for 6 h, stimulated with VEGF for 4 h, and immunoprecipitated with an AMIGO2 antibody and blotted with endogenous PDK1. (G) AMIGO2-deficient HUVECs were immunoprecipitated with an AMIGO2 antibody and blotted with an anti-PDK1 antibody. (H) Flag-PDK1 was immunoprecipitated and blotted with an anti-GFP antibody tagged to AMIGO2. A2, AMIGO2; HC, heavy chain; IB, immunoblotting; IgG, normal IgG; IP, immunoprecipitation; ns, not significant; WCL, whole cell lysate.