full length human amigo2 (Genecopoeia)
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full length human amigo2
Full Length Human Amigo2, supplied by Genecopoeia, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/amigo2/ORF+expression+clone+for+AMIGO2/pmc04639856-202-0-6
Average 94 stars, based on 2 article reviews
Full Length Human Amigo2, supplied by Genecopoeia, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/amigo2/ORF+expression+clone+for+AMIGO2/pmc04639856-202-0-6
Average 94 stars, based on 2 article reviews
full length human amigo2 - by Bioz Stars,
2026-10
94/100 stars
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1) Product Images from "AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation"
Article Title: AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation
Journal: The Journal of Cell Biology
doi: 10.1083/jcb.201503113
Figure Legend Snippet: AMIGO2 regulates EC viability and angiogenesis. (A–J) HUVECs were pretransfected with scrambled siRNA and AMIGO2-specific siRNA and harvested for analysis 48 h after transfection. (A) FAK immunostaining was performed in parallel. Images on the right are enlargements of the boxed regions on the left. Bars, 20 µm. (B) Cell viability of AMIGO2 siRNA–transfected HUVECs in complete media and under conditions of serum-free starvation with or without VEGF ( n = 6). Data were collected from independent experiments and analyzed using a two-tailed unpaired t test. Data are means ± SD. (C) Expression of active caspase-3 and FAK fragments in AMIGO2 siRNA–transfected ECs. (D) Different stages of apoptosis were detected by phycoerythrin-conjugated annexin V flow cytometry and PerCP–7-AAD staining. Quadrant gates are based on unstained, annexin V, or 7-AAD alone. Quadrant regions show the percentage of living cells (7-AAD − /annexin V − ), early apoptotic cells (7-AAD − /annexin V + ), late apoptotic cells (7-AAD + /annexin V + ), and necrotic cells (7-AAD + /annexin V − ). The data are representative of three experiments conducted by using different samples. (E and F) Wound healing migration and gelatin-coated Transwell migrations with or without VEGF were performed. Two types of siA2 were evaluated for wound healing migration. n = 3 and n = 6. (G) Matrigel-induced tube formation by AMIGO2 siRNA–transfected HUVECs was assessed. (H and J) Mean numbers of tubule branch points and of tubes per field in randomly selected images were quantified. (I) Tube length is presented as the percentage of total tube length per field versus that of untreated control cells. (H–J) Data were collected from independent experiments and analyzed using a two-tailed unpaired t test. Data are means ± SD. n = 4. *, P < 0.05; **, P < 0.005; ***, P < 0.0001. sc, scrambled siRNA; siA2, AMIGO2 siRNA.
Techniques Used: Transfection, Immunostaining, Two Tailed Test, Expressing, Flow Cytometry, Staining, Migration, Control
Figure Legend Snippet: AMIGO2 is required for angiogenesis in vivo. P3.5 mice were injected with Amigo2 -specific or control siRNA and shRNA, and experiments were performed at P5.5, P8.5, and P15.5. (A) Impairment of hyaloid vessel structures in Amigo2 siRNA–injected mice. Bars, 200 µm. (B) Capillary quantification. n = 5. (C) Hyaloid vessel staining with CD31, AMIGO2, and TUNEL was performed in Amigo2 shRNA–injected P5.5 mice. (D and E) Visualization of blood vessels by iB4 staining with AMIGO2 and TUNEL staining of Amigo2 -depleted retinas from P5.5 mice. Bars: (D) 50 µm; (E, left to right) 500 µm, 200 µm, and 50 µm. (E) Middle and right images are enlargements of the left image. (F–H) Radial expansions, retinal vascular branch points, and sprouts per field were quantified. n = 5. (I–L) Immunostaining of blood vessels by iB4-staining of Amigo2 shRNA–injected P8.5 retinas (I and K) and P15.5 retinas (J and L). Superficial (S), intermediate (I), and deep (D) layers of retinal vessels were taken and quantified. Bars, 50 µm. n = 5. Data were collected from mice ( n = 5–6) and analyzed using a two-tailed unpaired t test. *, P < 0.05; **, P < 0.005; ***, P < 0.0001. Data are means ± SD. shCon, control shRNA; shA2, Amigo2 shRNA.
Techniques Used: In Vivo, Injection, Control, shRNA, Staining, TUNEL Assay, Immunostaining, Two Tailed Test
Figure Legend Snippet: AMIGO2 modulates the PDK1–Akt pathway. (A) The endogenous effect of AMIGO2 knockdown on Akt signaling in HUVECs. Two types of siA2 were evaluated. (B) Endogenous effects of AMIGO overexpression on Akt signaling activation in HUVECs. (C and D) Akt phosphorylation in hyaloid vessels (C) and retinas (D) from Amigo2 -depleted mice was evaluated. The data are representative of six mice. (E) Temporal effects of AMIGO2 knockdown on VEGF-induced signaling pathways (20 ng/ml) in HUVECs. (F) VEGF-induced PI3K–PDK1–Akt signaling was evaluated in AMIGO2 siRNA–transfected HUVECs.
Techniques Used: Knockdown, Over Expression, Activation Assay, Phospho-proteomics, Protein-Protein interactions, Transfection
Figure Legend Snippet: 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.
Techniques Used: Immunostaining, Membrane, Cell Fractionation, Western Blot, Control, Immunoprecipitation, Clinical Proteomics
Figure Legend Snippet: The PH domain of PDK1 binds to the CD of AMIGO2. (A and B) The CD of AMIGO2 interacts with PDK1. HEK293 cells were transfected with Flag-PDK1 and GFP-tagged AMIGO2 or domain deletion mutants. Lysates were immunoprecipitated with GFP (A) or Flag (B) antibodies and blotted with the GFP or Flag antibody. PDK1 was immunoprecipitated with the Flag antibody and detected with the anti-GFP antibody. A2, AMIGO2 WT ; ΔLRR, AMIGO2 ΔLRR ; ΔIgG, AMIGO2 ΔIgG ; ΔCD, AMIGO2 ΔCD ; CD, CD domain of AMIGO2. (C) Schematic of 491 aa and 457 aa of the AMIGO2 constructs. (D) PDK1 associates with the region between 491 aa and 457 aa of AMIGO2. HEK293 cells were transfected with Flag-PDK1– and GFP-tagged AMIGO2 or domain deletion mutants. Lysates were immunoprecipitated with GFP antibody and blotted with the Flag and GFP antibodies. (E) Each His-tagged PDK1 kinase and His-tagged PDK1 PH was incubated with purified GST-AMIGO2 CD . Mixtures of His-tagged PDK1 kinase or PDK1 PH proteins and GST-AMIGO2 CD proteins were pulled down with GST resin and analyzed by Western blotting using anti-His and -GST antibodies. His-kinase, His-PDK1 kinase ; His-PH, His-PDK1 PH ; GST-CD, GST-AMIGO2 CD . (F) Mixtures of His-PDK1 PH and GST-CD proteins were incubated with PIP3-coated or control beads and treated with DM-PIT-1. The PIP3-bound proteins were analyzed with His and GST antibodies. (G) Multiple sequence alignments of the AMIGO family CD domains were performed. An asterisk indicates that the alignment contains identical amino acid residues in all sequences (or identical bases if DNA sequences are aligned); a colon indicates that the alignment contains different but highly conserved (very similar) amino acids; a period indicates that the alignment contains different amino acids that are somewhat similar; and a blank space indicates that the alignment contains dissimilar amino acids or gaps (or different bases if DNA sequences are aligned). (H) Diagram showing the protein sequences of cytosolic-truncated mutants. The total length of the cytosolic tail is 103 aa. (I) Far-western analysis was performed for each truncated mutant. Coomassie blue staining revealed purified GST-tagged truncated mutants that were previously loaded for far-western analysis. (J) Mixtures of purified GST-tagged truncated mutants and His-tagged PH domain proteins were pulled down with GST beads and eluted, and the Western blot was performed using His and GST antibodies. HC, heavy chain; IP, immunoprecipitation.
Techniques Used: Transfection, Immunoprecipitation, Construct, Incubation, Purification, Western Blot, Control, Sequencing, Mutagenesis, Staining
Figure Legend Snippet: PTD-A2, an AMIGO2 competitive peptide, blocks PDK1 translocation and Akt activation. (A) The TAT-A2 binding assay was performed. PH domain proteins (0–60 µg) were incubated with 1-µM FITC–TAT-A2 and detected by fluorescence. Data were collected from independent experiments and analyzed using a two-tailed unpaired t test. n = 4. (B) Peptide competition assay with Con or TAT-A2 and CD to PH domains. His-tagged PDK1 PH was incubated with purified GST-AMIGO2 CD and Con or TAT-A2 in a dose-dependent manner. His-tagged PH domain proteins were pulled down with GST resins, eluted, and analyzed by Western blotting. (C) Con- and TAT-A2– treated HUVECs were immunoprecipitated with an AMIGO2 antibody and blotted with anti-PDK1 and AMIGO2 antibodies. (D) Localization of PDK1 in the plasma membrane and cytosol of HUVECs in the presence of VEGF after Con or TAT-A2 treatment. Bars, 20 µm. (E) Subcellular localization analysis was performed by cell fractionation in VEGF-treated, TAT-A2–treated HUVECs. (F) Quantification of Western blots. Subcellular localization analysis was performed by cell fractionation with or without VEGF in control and TAT-A2 HUVECs. HUVECs were affected by both PTD-A2 peptides. (G) Effects of TAT-A2 on VEGF-induced Akt signaling in HUVECs. Con, control peptide; TAT-A2, TAT-RVVFLEPLKD peptide. PTD-A2 means both TAT-A2 and NGR-A2. Data are means ± SD. *, P < 0.05; **, P < 0.005; ***, P < 0.0001. HC, heavy chain; IB, immunoblotting; IP, immunoprecipitation; WCL, whole cell lysate.
Techniques Used: Translocation Assay, Activation Assay, Binding Assay, Incubation, Fluorescence, Two Tailed Test, Competitive Binding Assay, Purification, Western Blot, Immunoprecipitation, Clinical Proteomics, Membrane, Cell Fractionation, Control
Figure Legend Snippet: Treatment with PTD-A2 inhibits tumor growth and tumor angiogenesis. (A–E) Treatment with FITC–NGR-A2 inhibits tumor growth, neovessel formation, and tumor cell death in B16F10 tumors. (A) Photograph of B16F10 melanoma tumors resected after 13 d. Bar, 5 mm. (B) Comparison of tumor volume ( n = 7 mice per group; 4 mg/kg NGR-Con or NGR-A2 was administered every other day). (C–E) Images (C) and quantification (D) of CD31-positive blood vessels (red) and TUNEL-positive cells (pink) over CD31-positive blood vessels (E) in B16F10 melanoma tumors. FITC, green; DAPI, blue. Bars, 50 µm. (F–I) Amigo2 shRNA–transfected B16F10 tumors with NGR-A2 display dramatically inhibited tumor growth and tumor vessel formation. (F) Image of Amigo2 shRNA–transfected B16F10 melanoma tumors resected after 14 d. Bar, 5 mm. (G) Comparison of tumor volumes ( n = 7–8 mice per group; 4 mg/kg NGR-Con or NGR-A2 was administered every other day). (H and I) Images (H) and quantification (I) of CD31-positive blood vessels (red). (B and G) Data were analyzed using repeated measures of two-way analysis of variance. *, P < 0.05; **, P < 0.005; ***, P < 0.0001. ns, not significant. Data are means ± SD.
Techniques Used: Comparison, TUNEL Assay, shRNA, Transfection
Figure Legend Snippet: The AMIGO2–PDK1–Akt signaling pathway. (A) This model proposes that the direct binding of AMIGO2 to the PDK1 PH domain enhanced the activation of PDK1 and Akt in the plasma membrane, which resulted in increased EC viability, adhesion, migration, and angiogenesis. (B) Loss of AMIGO2 attenuated the phosphorylation of PDK1 and Akt and EC survival and adhesion. (C) PTD-A2, a specific peptide in the C-terminal region of AMIGO2, effectively abrogated the phosphorylation of PDK1 and Akt, cell survival, OIR-induced angiogenesis, tumor angiogenesis, and tumor growth. P, phosphorylation.
Techniques Used: Binding Assay, Activation Assay, Clinical Proteomics, Membrane, Migration, Phospho-proteomics
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