Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: Increased HSD11β1 Expression in Human Leiomyomatous Uteri: Implication for Enhanced Glucocorticoid Signaling

doi: 10.1210/clinem/dgaf255

Figure Lengend Snippet: Silencing of FKBP5 blunts dexamethasone (DEX)-regulated expression of glucocorticoid response element-containing genes in primary leiomyoma cell cultures. qPCR analysis of mRNA levels for DEX-upregulated genes TSC22D 3 , ADH1B, IL1R1 (A), and DEX-downregulated genes GREM1, IGFBP1, AMIGO2 (B) in cultured leiomyoma cells transfected with either control or FKBP5 siRNA for 48 hours and then treated with either vehicle (Cont) or DEX for 24 hours. Bars represent mean ± standard error of the mean, n = 4.

Article Snippet: The full name and Taq-Man Probe Assay IDs for each target gene include: FK506-binding protein 5 ( FKBP5 )- Hs01561006_m1; Hydroxysteroid 11-β dehydrogenase 1 ( HSD11B1) - Hs01547870_m1; IL-1 receptor type 1 ( IL1R1 )- Hs00991002_m1; TSC22 domain family member 3 ( TSC22D3 )- Hs00608272_m1; alcohol dehydrogenase 1B, β polypeptide ( ADH1B )- Hs00605175_m1; Gremlin 1, DAN family BMP antagonist ( GREM1 )- Hs00171951_m1; IGF-binding protein 5 ( IGFBP5 )- Hs00181213_m1; adhesion molecule with Ig-like domain 2 ( AMIGO2 )- Hs05001325_s1; laminin subunit alpha 2 ( LAMA2 )- Hs00166308_m1; fibronectin 1 ( FN1 )- Hs00365052_m1; calponin 1 ( CNN1 )- Hs00959434_m1; actin β ( ACTB )- Hs99999903_m1; and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH )- Hs99999905_m1.

Techniques: Expressing, Cell Culture, Transfection, Control

Journal: NPJ Precision Oncology

Article Title: Metabolomic and transcriptomic profiling of HNSCC identifies AMIGO2 as a therapeutic target modulating tumor microenvironment

doi: 10.1038/s41698-025-01132-z

Figure Lengend Snippet: a Expression of AMIGO2, STC2, INHBA, DKK1, VEGFC, SPOCK1, MT1E, and FOXD1 in scRNA-seq UMAP plots. b Bar plot showing cell type proportions under LRS subtypes from scRNA-seq data. c AMIGO2, ranked highly for prognostic importance, was selected for further analysis. d AMIGO2 expression is upregulated in most tumor tissues across various cancers in TCGA pan-cancer dataset. e Evaluation of AMIGO2’s prognostic value (OS) in TCGA pan-cancer dataset. f Correlation analysis between AMIGO2 expression and representative signatures in TCGA pan-cancer dataset. Ns: no significant difference, * P < 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Article Snippet: The following primary antibodies were used for Western blotting: AMIGO2 (1:500, HUAAN, catalog No: ER1903-66), GAPDH (1:1000, CST, catalog No: 5174S, RRID: AB_10622025), E-cadherin (1:1000, CST, catalog No: 3195S, RRID: AB_2291471), N-cadherin (1:1000, CST, catalog No: 13116S, RRID: AB_2687616), HPRT1 (1:1000, Proteintech Cat# 15059-1-AP, RRID: AB_10638622), PAICS (1:1000, Proteintech Cat# 12967-1-AP, RRID: AB_10638449), Vimentin (1:1000, CST, catalog No: 5741S, RRID: AB_10695459), MMP9 (1:1000, CST, catalog No: 13667S, RRID: AB_2798289), FAP (Thermo Fisher Scientific Cat# PA5-99313, RRID: AB_2818246), α-SMA (Cell Signaling Technology Cat# 19245, RRID: AB_2734735), PI3K (Cell Signaling Technology Cat# 4292, RRID:AB_329869), Phospho-PI3K (Cell Signaling Technology Cat# 17366, RRID:AB_2895293), AKT (Cell Signaling Technology Cat# 9272, RRID: AB_329827), Phospho-Akt (Thr308) (Cell Signaling Technology Cat# 9275, RRID: AB_329828), Phospho-Akt (Ser473) (Cell Signaling Technology Cat# 9271, RRID: AB_329825), PDK1 (Thermo Fisher Scientific Cat# A302-130A, RRID:AB_1720395), Phospho-PDK1 (Ser241) (Cell Signaling Technology Cat# 3061, RRID: AB_2161919), mTOR (Abcam Cat# ab134903, RRID: AB_2800465) and mTOR (phospho S2481) (Abcam Cat# ab137133, RRID:AB_2800466).

Techniques: Expressing

Journal: NPJ Precision Oncology

Article Title: Metabolomic and transcriptomic profiling of HNSCC identifies AMIGO2 as a therapeutic target modulating tumor microenvironment

doi: 10.1038/s41698-025-01132-z

Figure Lengend Snippet: a IHC images and quantitative statistics of AMIGO2 expression in adjacent normal tissue, precancerous lesions, and tumor tissues. b Representative images and quantification of CD8, CD56, FAP, and PanCK staining in tumor tissues from high-AMIGO2 and low-AMIGO2 patient groups. c Graphical abstract for this study. Ns: no significant difference. * P < 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Article Snippet: The following primary antibodies were used for Western blotting: AMIGO2 (1:500, HUAAN, catalog No: ER1903-66), GAPDH (1:1000, CST, catalog No: 5174S, RRID: AB_10622025), E-cadherin (1:1000, CST, catalog No: 3195S, RRID: AB_2291471), N-cadherin (1:1000, CST, catalog No: 13116S, RRID: AB_2687616), HPRT1 (1:1000, Proteintech Cat# 15059-1-AP, RRID: AB_10638622), PAICS (1:1000, Proteintech Cat# 12967-1-AP, RRID: AB_10638449), Vimentin (1:1000, CST, catalog No: 5741S, RRID: AB_10695459), MMP9 (1:1000, CST, catalog No: 13667S, RRID: AB_2798289), FAP (Thermo Fisher Scientific Cat# PA5-99313, RRID: AB_2818246), α-SMA (Cell Signaling Technology Cat# 19245, RRID: AB_2734735), PI3K (Cell Signaling Technology Cat# 4292, RRID:AB_329869), Phospho-PI3K (Cell Signaling Technology Cat# 17366, RRID:AB_2895293), AKT (Cell Signaling Technology Cat# 9272, RRID: AB_329827), Phospho-Akt (Thr308) (Cell Signaling Technology Cat# 9275, RRID: AB_329828), Phospho-Akt (Ser473) (Cell Signaling Technology Cat# 9271, RRID: AB_329825), PDK1 (Thermo Fisher Scientific Cat# A302-130A, RRID:AB_1720395), Phospho-PDK1 (Ser241) (Cell Signaling Technology Cat# 3061, RRID: AB_2161919), mTOR (Abcam Cat# ab134903, RRID: AB_2800465) and mTOR (phospho S2481) (Abcam Cat# ab137133, RRID:AB_2800466).

Techniques: Expressing, Staining

Journal: PLoS ONE

Article Title: AMIGO3 Is an NgR1/p75 Co-Receptor Signalling Axon Growth Inhibition in the Acute Phase of Adult Central Nervous System Injury

doi: 10.1371/journal.pone.0061878

Figure Lengend Snippet: Microarray analysis to show fold-differences in mRNA levels compared to intact controls of described genes in rat DRG 1 d after DC lesion.

Article Snippet: Primary antibodies included: goat anti-human AMIGO, AMIGO2, AMIGO3 and TROY (all used at 1∶100 dilution and from Santa Cruz Biotechnology, Santa Cruz, CA, USA); monoclonal anti-NGFR (1∶200 dilution; Sigma); rabbit anti-NGFR1 (1∶200 dilution; Alpha Diagnostics, San Antonio, TX, USA); and rabbit anti-LINGO1 (1∶400 dilution; Abcam, Cambridge, UK).

Techniques: Microarray

Journal: PLoS ONE

Article Title: AMIGO3 Is an NgR1/p75 Co-Receptor Signalling Axon Growth Inhibition in the Acute Phase of Adult Central Nervous System Injury

doi: 10.1371/journal.pone.0061878

Figure Lengend Snippet: ( A ) Semi-quantitative RT-PCR and ( B ) densitometry to show changes in mRNA levels of AMIGO isoforms and LINGO-1 expressed in non-regenerating DRGN compared to intact controls. GAPDH is used as a housekeeping gene. ( C ) Representative western blots and ( D ) densitometry to show changes in protein levels of AMIGO isoforms and LINGO-1 in non-regenerating DRGN relative to intact controls. ( E–H ) Immunohistochemistry in sections from intact controls and 1 d DC injured DRGN shows immunolabelling for AMIGO ( E ), AMIGO2 ( F ), AMIGO3 ( G ) and ( H ) LINGO-1. Insets in E-H show images from blocking peptide controls to demonstrate specificity of the relevant antibody Scale bars in E–H = 100 µm. *** = P<0.0001, ANOVA.

Article Snippet: Primary antibodies included: goat anti-human AMIGO, AMIGO2, AMIGO3 and TROY (all used at 1∶100 dilution and from Santa Cruz Biotechnology, Santa Cruz, CA, USA); monoclonal anti-NGFR (1∶200 dilution; Sigma); rabbit anti-NGFR1 (1∶200 dilution; Alpha Diagnostics, San Antonio, TX, USA); and rabbit anti-LINGO1 (1∶400 dilution; Abcam, Cambridge, UK).

Techniques: Quantitative RT-PCR, Western Blot, Immunohistochemistry, Blocking Assay

Journal: PLoS ONE

Article Title: AMIGO3 Is an NgR1/p75 Co-Receptor Signalling Axon Growth Inhibition in the Acute Phase of Adult Central Nervous System Injury

doi: 10.1371/journal.pone.0061878

Figure Lengend Snippet: Microarray analysis to show fold-differences in mRNA levels of described genes compared to intact controls in rat DRG 10 d after DC lesion, sciatic nerve (SN) or pre-conditioning (pSN+DC) lesions.

Article Snippet: Primary antibodies included: goat anti-human AMIGO, AMIGO2, AMIGO3 and TROY (all used at 1∶100 dilution and from Santa Cruz Biotechnology, Santa Cruz, CA, USA); monoclonal anti-NGFR (1∶200 dilution; Sigma); rabbit anti-NGFR1 (1∶200 dilution; Alpha Diagnostics, San Antonio, TX, USA); and rabbit anti-LINGO1 (1∶400 dilution; Abcam, Cambridge, UK).

Techniques: Microarray

Journal: PLoS ONE

Article Title: AMIGO3 Is an NgR1/p75 Co-Receptor Signalling Axon Growth Inhibition in the Acute Phase of Adult Central Nervous System Injury

doi: 10.1371/journal.pone.0061878

Figure Lengend Snippet: ( A ) Representative AMIGO3 localisation in DRGN (arrows = AMIGO3 + , arrowhead = AMIGO3 − ) and ( B ) quantification of the percentage ± SEM of DRGN which are AMIGO3 + and AMIGO3 − , in DRG cultures. ( C ) Western blotting and densitometry showing the levels of knockdown, compared to Scr-siAMIGO3 and siGFP after siAMIGO3 treatment. Re-stripped western blots are also shown for AMIGO, AMIGO2 and LINGO-1 to demonstrate that knockdown of AMIGO3 does not affect the levels of these molecules in culture. β-actin is used as a loading control. ( D ) Rho activation assay in untransfected and siRNA transfected DRG cells in the presence of CME. Total Rho levels were also detected by western blotting. Scale bar in (A) = 50 µm.

Article Snippet: Primary antibodies included: goat anti-human AMIGO, AMIGO2, AMIGO3 and TROY (all used at 1∶100 dilution and from Santa Cruz Biotechnology, Santa Cruz, CA, USA); monoclonal anti-NGFR (1∶200 dilution; Sigma); rabbit anti-NGFR1 (1∶200 dilution; Alpha Diagnostics, San Antonio, TX, USA); and rabbit anti-LINGO1 (1∶400 dilution; Abcam, Cambridge, UK).

Techniques: Western Blot, Knockdown, Control, Activation Assay, Transfection

Journal: The Journal of Cell Biology

Article Title: AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation

doi: 10.1083/jcb.201503113

Figure Lengend 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.

Article Snippet: Mouse and goat anti-AMIGO2 antibodies for immunoprecipitation and Western blotting were purchased from R&D Systems and Santa Cruz Biotechnology, Inc., respectively.

Techniques: Transfection, Immunostaining, Two Tailed Test, Expressing, Flow Cytometry, Staining, Migration, Control

Journal: The Journal of Cell Biology

Article Title: AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation

doi: 10.1083/jcb.201503113

Figure Lengend 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.

Article Snippet: Mouse and goat anti-AMIGO2 antibodies for immunoprecipitation and Western blotting were purchased from R&D Systems and Santa Cruz Biotechnology, Inc., respectively.

Techniques: In Vivo, Injection, Control, shRNA, Staining, TUNEL Assay, Immunostaining, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation

doi: 10.1083/jcb.201503113

Figure Lengend 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.

Article Snippet: Mouse and goat anti-AMIGO2 antibodies for immunoprecipitation and Western blotting were purchased from R&D Systems and Santa Cruz Biotechnology, Inc., respectively.

Techniques: Knockdown, Over Expression, Activation Assay, Phospho-proteomics, Protein-Protein interactions, Transfection

Journal: The Journal of Cell Biology

Article Title: AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation

doi: 10.1083/jcb.201503113

Figure Lengend 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.

Article Snippet: Mouse and goat anti-AMIGO2 antibodies for immunoprecipitation and Western blotting were purchased from R&D Systems and Santa Cruz Biotechnology, Inc., respectively.

Techniques: Immunostaining, Membrane, Cell Fractionation, Western Blot, Control, Immunoprecipitation, Clinical Proteomics

Journal: The Journal of Cell Biology

Article Title: AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation

doi: 10.1083/jcb.201503113

Figure Lengend 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.

Article Snippet: Mouse and goat anti-AMIGO2 antibodies for immunoprecipitation and Western blotting were purchased from R&D Systems and Santa Cruz Biotechnology, Inc., respectively.

Techniques: Transfection, Immunoprecipitation, Construct, Incubation, Purification, Western Blot, Control, Sequencing, Mutagenesis, Staining

Journal: The Journal of Cell Biology

Article Title: AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation

doi: 10.1083/jcb.201503113

Figure Lengend 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.

Article Snippet: Mouse and goat anti-AMIGO2 antibodies for immunoprecipitation and Western blotting were purchased from R&D Systems and Santa Cruz Biotechnology, Inc., respectively.

Techniques: Translocation Assay, Activation Assay, Binding Assay, Incubation, Fluorescence, Two Tailed Test, Competitive Binding Assay, Purification, Western Blot, Immunoprecipitation, Clinical Proteomics, Membrane, Cell Fractionation, Control

Journal: The Journal of Cell Biology

Article Title: AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation

doi: 10.1083/jcb.201503113

Figure Lengend 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.

Article Snippet: Mouse and goat anti-AMIGO2 antibodies for immunoprecipitation and Western blotting were purchased from R&D Systems and Santa Cruz Biotechnology, Inc., respectively.

Techniques: Comparison, TUNEL Assay, shRNA, Transfection

Journal: The Journal of Cell Biology

Article Title: AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation

doi: 10.1083/jcb.201503113

Figure Lengend 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.

Article Snippet: Mouse and goat anti-AMIGO2 antibodies for immunoprecipitation and Western blotting were purchased from R&D Systems and Santa Cruz Biotechnology, Inc., respectively.

Techniques: Binding Assay, Activation Assay, Clinical Proteomics, Membrane, Migration, Phospho-proteomics

Journal: International Journal of Medical Sciences

Article Title: AMIGO2 as a Novel Biomarker Predicting Poor Prognosis and Associated with Adhesion-Driven Metastasis in Pancreatic Adenocarcinoma

doi: 10.7150/ijms.121794

Figure Lengend Snippet: Transcriptional expression and pathological stage analysis of AMIGO family members in PAAD using GEPIA2 database. (A-B) Comparison of AMIGO1, AMIGO2 and AMIGO3 mRNA expression levels between PAAD and normal pancreatic tissues. (C) Expression levels of AMIGOs across different tumor stages in PAAD patients.

Article Snippet: Primary antibodies used in this study included: AMIGO2 (E-AB-10790, Elabscience), E-cadherin (R55-180, BD Biosciences), Vimentin (E-AB-22085, Elabscience), and α-Tubulin (T6199, Sigma-Aldrich).

Techniques: Expressing, Comparison

Journal: International Journal of Medical Sciences

Article Title: AMIGO2 as a Novel Biomarker Predicting Poor Prognosis and Associated with Adhesion-Driven Metastasis in Pancreatic Adenocarcinoma

doi: 10.7150/ijms.121794

Figure Lengend Snippet: Prognostic value of AMIGO family in pancreatic adenocarcinoma (PAAD) patients. (A) Relapse-free survival (RFS) curves and (B) Overall survival (OS) of AMIGO1, AMIGO2 and AMIGO3 in PAAD.

Article Snippet: Primary antibodies used in this study included: AMIGO2 (E-AB-10790, Elabscience), E-cadherin (R55-180, BD Biosciences), Vimentin (E-AB-22085, Elabscience), and α-Tubulin (T6199, Sigma-Aldrich).

Techniques:

Journal: International Journal of Medical Sciences

Article Title: AMIGO2 as a Novel Biomarker Predicting Poor Prognosis and Associated with Adhesion-Driven Metastasis in Pancreatic Adenocarcinoma

doi: 10.7150/ijms.121794

Figure Lengend Snippet: Genetic alterations, gene and protein interaction networks and co-expression analysis of AMIGO family members in pancreatic adenocarcinoma (PAAD). (A-B) Genetic alteration analysis of AMIGO1, AMIGO2 and AMIGO3 in PAAD patients using cBioPortal. (C) Gene-gene interaction (GGI) network of AMIGOs constructed by GeneMANIA. (D) Protein-protein interaction (PPI) network of AMIGOs generated using the STRING database. (E) Co-expression gene analysis of AMIGOs based on the intersection of top 1000 correlated genes, visualized using VENNY.

Article Snippet: Primary antibodies used in this study included: AMIGO2 (E-AB-10790, Elabscience), E-cadherin (R55-180, BD Biosciences), Vimentin (E-AB-22085, Elabscience), and α-Tubulin (T6199, Sigma-Aldrich).

Techniques: Expressing, Construct, Generated

Journal: International Journal of Medical Sciences

Article Title: AMIGO2 as a Novel Biomarker Predicting Poor Prognosis and Associated with Adhesion-Driven Metastasis in Pancreatic Adenocarcinoma

doi: 10.7150/ijms.121794

Figure Lengend Snippet: Functional enrichment analysis of AMIGO2 in pancreatic adenocarcinoma (PAAD) by using the DAVID platform. (A-C) Gene Ontology (GO) enrichment analysis of AMIGO2-related genes, categorized into biological processes (BP), cellular components (CC), and molecular functions (MF), respectively. (D) Top 20 GO terms enriched in AMIGO2 co-expressed genes. (E) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of AMIGO2-associated genes.

Article Snippet: Primary antibodies used in this study included: AMIGO2 (E-AB-10790, Elabscience), E-cadherin (R55-180, BD Biosciences), Vimentin (E-AB-22085, Elabscience), and α-Tubulin (T6199, Sigma-Aldrich).

Techniques: Functional Assay

Journal: International Journal of Medical Sciences

Article Title: AMIGO2 as a Novel Biomarker Predicting Poor Prognosis and Associated with Adhesion-Driven Metastasis in Pancreatic Adenocarcinoma

doi: 10.7150/ijms.121794

Figure Lengend Snippet: Correlation between AMIGO2 and EMT-related transcription factors in PAAD. (A) Protein expression of AMIGO2 in normal pancreatic tissues and PAAD tissues based on immunohistochemistry (IHC) images from the Human Protein Atlas (HPA). Representative IHC staining shows weak or absent AMIGO2 expression in normal pancreatic tissues (left) and stronger staining intensity in PAAD tissues (right). (B-G) Correlation analyses between AMIGO2 and key EMT-related transcription factors in TCGA-PAAD. Scatter plots show the Spearman correlation between AMIGO2 expression and ZEB1 (ρ = 0.27, p = 2.7 × 10⁻⁴), SNAI1 (ρ = 0.23, p = 1.8 × 10⁻³), SP1 (ρ = 0.40, p = 3.6 × 10⁻⁸), NFKB1 (ρ = 0.22, p = 3.8 × 10⁻³), RELA (ρ = 0.41, p = 1.8 × 10⁻⁸) and TWIST1 (ρ = 0.37, p = 4.1 × 10⁻⁷). (H) Correlation between AMIGO2 expression and immune cell infiltration across TCGA cancer types, including pancreatic adenocarcinoma (PAAD), pheochromocytoma and paraganglioma (PCPG), and prostate adenocarcinoma (PRAD), analyzed using TIMER2.0 with tumor purity adjustment. The heatmap displays partial Spearman correlation coefficients across multiple immune deconvolution algorithms. (I) Correlation between AMIGO2 and CD274 (PD-L1) expression in TCGA-PAAD, analyzed using GEPIA2 (Spearman correlation). Scatter plot shows a significant positive correlation (R = 0.34, p = 3.9 × 10⁻⁶), indicating that AMIGO2-high tumors exhibit elevated PD-L1 expression.

Article Snippet: Primary antibodies used in this study included: AMIGO2 (E-AB-10790, Elabscience), E-cadherin (R55-180, BD Biosciences), Vimentin (E-AB-22085, Elabscience), and α-Tubulin (T6199, Sigma-Aldrich).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: International Journal of Medical Sciences

Article Title: AMIGO2 as a Novel Biomarker Predicting Poor Prognosis and Associated with Adhesion-Driven Metastasis in Pancreatic Adenocarcinoma

doi: 10.7150/ijms.121794

Figure Lengend Snippet: AMIGO2 promotes migration and invasion by inducing EMT in pancreatic cancer cells. (A) Western blot analysis of AMIGO2 protein levels in MiaPaCa-2 cells transfected with two independent AMIGO2 shRNAs (kd-AMIGO2 A1 and kd-AMIGO2 B2) compared with control cells. α-Tubulin served as a loading control. (B) Western blot analysis of EMT markers showing that AMIGO2 knockdown upregulated E-cadherin and downregulated Vimentin expression. α-Tubulin was used as an internal control. (C) Representative images of wound-healing assays performed in MiaPaCa-2 cells after AMIGO2 knockdown, captured at 0, 24, and 48 h. (D) Quantitative analysis of wound closure rates (%) in MiaPaCa-2 control and AMIGO2-silenced cells. (E) Representative images of Transwell invasion assays showing a reduced number of invading cells following AMIGO2 knockdown. (F) Statistical quantification of invaded cell area (%) based on Transwell assays. ***, p<0.01.

Article Snippet: Primary antibodies used in this study included: AMIGO2 (E-AB-10790, Elabscience), E-cadherin (R55-180, BD Biosciences), Vimentin (E-AB-22085, Elabscience), and α-Tubulin (T6199, Sigma-Aldrich).

Techniques: Migration, Western Blot, Transfection, Control, Knockdown, Expressing

Journal: Molecular cell

Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

doi: 10.1016/j.molcel.2017.11.004

Figure Lengend Snippet: (A) AMIGO2 expression levels detected by qRT-PCR in NHM and a panel of metastatic melanoma cell lines harboring distinct melanoma mutations. Data are normalized to GAPDH.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies BRD2 Bethyl Laboratories A302-583A BRD4 Bethyl Laboratories A301-985A H3K4me1 Abcam ab8895 H3K4me3 Abcam ab1012 H3K27ac Abcam ab177178 H3K27me3 EMD 07-449 MED1 Bethyl Laboratories A300-793A macroH2A2 Bernstein Laboratory, ISMMS in house macroH2A1 Abcam ab37264 CTCF EMD 07-729 FOSL2 Santa Cruz SC-604 TEAD4 Santa Cruz SC-101184 HSP90 Santa Cruz SC-13119 GAPDH Santa Cruz SC-32233 GFP Roche 11814460001 LAMIN B1 Santa Cruz SC-6217 ALCAM Abcam ab109215 L1CAM Abcam ab20148 NRP2 Santa Cruz SC-13117 NRP1 Abcam ab81321 TUBULIN Sigma T9026 FOXM1 Cell Signaling Technology 5436 Casp3 Cell Signaling Technology 9662 PARP Cell Signaling Technology 9542 AMIGO2 (for IHC) Abnova H00347902-B01P Lot# F8271 RRID:AB_1672842 AMIGO2 (for IF and WB) R&D systems AF2080 PTK7 (N-terminal) (for WB) R&D systems AF4499 PTK7 (C-terminal) (for IF and WB) Cell Signaling 11926 Secondary rabbit anti goat (for AMIGO2 detection on WB) Sigma A5420 HRP-conjugated ACTIN Sigma A3854 Biological Samples Melanoma and normal skin tissue microarray Biomax ME208 Nevus tissue microarray IMCG, NYU MT032808 Patient derived short term cultures Osman Laboratory, NYU de Miera et al., 2012 NHM (this paper) Bernstein Laboratory, ISMMS NHM1-5 Chemicals, Peptides, and Recombinant Proteins GFP Trap-A beads Chromotek gta-10 BET inhibitor JQ1 Bradner Laboratory; Agreement # {"type":"entrez-nucleotide","attrs":{"text":"A09608","term_id":"490581","term_text":"A09608"}} A09608 N/A BET inhibitor I-BET762 Apex Bio 501014747 IGEPAL Sigma I-8896 Micrococcal Nuclease Affymetrix 70196Y Dynabeads Protein A Immunoprécipitation kit Invitrogen 10006D Magna ChIP Protein A+G Magnetic beads Millipore 16-663 DSG Pierce (ThermoFisher) 20593 Critical Commercial Assays Click- Click-IT EdU Pacific Blue Flow Cytometry Assay Kit ThermoFisher {"type":"entrez-nucleotide","attrs":{"text":"C10636","term_id":"1535707","term_text":"C10636"}} C10636 Caspase Activity assay MBL Int.

Techniques: Expressing, Quantitative RT-PCR

Journal: Molecular cell

Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

doi: 10.1016/j.molcel.2017.11.004

Figure Lengend Snippet: (A) Relative growth curves of 501MEL and SKmel147 cells stably transduced with non-targeting scrambled control shRNA (shSCR) and two AMIGO2 shRNAs (shA2 #1 and #2). Values are normalized to seeding control (n = 3).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies BRD2 Bethyl Laboratories A302-583A BRD4 Bethyl Laboratories A301-985A H3K4me1 Abcam ab8895 H3K4me3 Abcam ab1012 H3K27ac Abcam ab177178 H3K27me3 EMD 07-449 MED1 Bethyl Laboratories A300-793A macroH2A2 Bernstein Laboratory, ISMMS in house macroH2A1 Abcam ab37264 CTCF EMD 07-729 FOSL2 Santa Cruz SC-604 TEAD4 Santa Cruz SC-101184 HSP90 Santa Cruz SC-13119 GAPDH Santa Cruz SC-32233 GFP Roche 11814460001 LAMIN B1 Santa Cruz SC-6217 ALCAM Abcam ab109215 L1CAM Abcam ab20148 NRP2 Santa Cruz SC-13117 NRP1 Abcam ab81321 TUBULIN Sigma T9026 FOXM1 Cell Signaling Technology 5436 Casp3 Cell Signaling Technology 9662 PARP Cell Signaling Technology 9542 AMIGO2 (for IHC) Abnova H00347902-B01P Lot# F8271 RRID:AB_1672842 AMIGO2 (for IF and WB) R&D systems AF2080 PTK7 (N-terminal) (for WB) R&D systems AF4499 PTK7 (C-terminal) (for IF and WB) Cell Signaling 11926 Secondary rabbit anti goat (for AMIGO2 detection on WB) Sigma A5420 HRP-conjugated ACTIN Sigma A3854 Biological Samples Melanoma and normal skin tissue microarray Biomax ME208 Nevus tissue microarray IMCG, NYU MT032808 Patient derived short term cultures Osman Laboratory, NYU de Miera et al., 2012 NHM (this paper) Bernstein Laboratory, ISMMS NHM1-5 Chemicals, Peptides, and Recombinant Proteins GFP Trap-A beads Chromotek gta-10 BET inhibitor JQ1 Bradner Laboratory; Agreement # {"type":"entrez-nucleotide","attrs":{"text":"A09608","term_id":"490581","term_text":"A09608"}} A09608 N/A BET inhibitor I-BET762 Apex Bio 501014747 IGEPAL Sigma I-8896 Micrococcal Nuclease Affymetrix 70196Y Dynabeads Protein A Immunoprécipitation kit Invitrogen 10006D Magna ChIP Protein A+G Magnetic beads Millipore 16-663 DSG Pierce (ThermoFisher) 20593 Critical Commercial Assays Click- Click-IT EdU Pacific Blue Flow Cytometry Assay Kit ThermoFisher {"type":"entrez-nucleotide","attrs":{"text":"C10636","term_id":"1535707","term_text":"C10636"}} C10636 Caspase Activity assay MBL Int.

Techniques: Stable Transfection, Transduction, shRNA

Journal: Molecular cell

Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

doi: 10.1016/j.molcel.2017.11.004

Figure Lengend Snippet: (A) Immunofluorescence of SKmel147 cells stably expressing AMIGO2-GFP (green), stained with AMIGO2 antibody (red) and Hoechst 33342 (blue). Scale bar, 20 μm.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies BRD2 Bethyl Laboratories A302-583A BRD4 Bethyl Laboratories A301-985A H3K4me1 Abcam ab8895 H3K4me3 Abcam ab1012 H3K27ac Abcam ab177178 H3K27me3 EMD 07-449 MED1 Bethyl Laboratories A300-793A macroH2A2 Bernstein Laboratory, ISMMS in house macroH2A1 Abcam ab37264 CTCF EMD 07-729 FOSL2 Santa Cruz SC-604 TEAD4 Santa Cruz SC-101184 HSP90 Santa Cruz SC-13119 GAPDH Santa Cruz SC-32233 GFP Roche 11814460001 LAMIN B1 Santa Cruz SC-6217 ALCAM Abcam ab109215 L1CAM Abcam ab20148 NRP2 Santa Cruz SC-13117 NRP1 Abcam ab81321 TUBULIN Sigma T9026 FOXM1 Cell Signaling Technology 5436 Casp3 Cell Signaling Technology 9662 PARP Cell Signaling Technology 9542 AMIGO2 (for IHC) Abnova H00347902-B01P Lot# F8271 RRID:AB_1672842 AMIGO2 (for IF and WB) R&D systems AF2080 PTK7 (N-terminal) (for WB) R&D systems AF4499 PTK7 (C-terminal) (for IF and WB) Cell Signaling 11926 Secondary rabbit anti goat (for AMIGO2 detection on WB) Sigma A5420 HRP-conjugated ACTIN Sigma A3854 Biological Samples Melanoma and normal skin tissue microarray Biomax ME208 Nevus tissue microarray IMCG, NYU MT032808 Patient derived short term cultures Osman Laboratory, NYU de Miera et al., 2012 NHM (this paper) Bernstein Laboratory, ISMMS NHM1-5 Chemicals, Peptides, and Recombinant Proteins GFP Trap-A beads Chromotek gta-10 BET inhibitor JQ1 Bradner Laboratory; Agreement # {"type":"entrez-nucleotide","attrs":{"text":"A09608","term_id":"490581","term_text":"A09608"}} A09608 N/A BET inhibitor I-BET762 Apex Bio 501014747 IGEPAL Sigma I-8896 Micrococcal Nuclease Affymetrix 70196Y Dynabeads Protein A Immunoprécipitation kit Invitrogen 10006D Magna ChIP Protein A+G Magnetic beads Millipore 16-663 DSG Pierce (ThermoFisher) 20593 Critical Commercial Assays Click- Click-IT EdU Pacific Blue Flow Cytometry Assay Kit ThermoFisher {"type":"entrez-nucleotide","attrs":{"text":"C10636","term_id":"1535707","term_text":"C10636"}} C10636 Caspase Activity assay MBL Int.

Techniques: Immunofluorescence, Stable Transfection, Expressing, Staining

Journal: Molecular cell

Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

doi: 10.1016/j.molcel.2017.11.004

Figure Lengend Snippet: (A) Capture of the UCSC genome browser (GRCh37/hg19) showing 800 kb region on human chromosome 12 (y axis = reads per kilobase per million reads). Ensembl genes (blue) and AMIGO2 SEs (red) are shown at the top. Putative TAD/LAD domain is shown at the bottom.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies BRD2 Bethyl Laboratories A302-583A BRD4 Bethyl Laboratories A301-985A H3K4me1 Abcam ab8895 H3K4me3 Abcam ab1012 H3K27ac Abcam ab177178 H3K27me3 EMD 07-449 MED1 Bethyl Laboratories A300-793A macroH2A2 Bernstein Laboratory, ISMMS in house macroH2A1 Abcam ab37264 CTCF EMD 07-729 FOSL2 Santa Cruz SC-604 TEAD4 Santa Cruz SC-101184 HSP90 Santa Cruz SC-13119 GAPDH Santa Cruz SC-32233 GFP Roche 11814460001 LAMIN B1 Santa Cruz SC-6217 ALCAM Abcam ab109215 L1CAM Abcam ab20148 NRP2 Santa Cruz SC-13117 NRP1 Abcam ab81321 TUBULIN Sigma T9026 FOXM1 Cell Signaling Technology 5436 Casp3 Cell Signaling Technology 9662 PARP Cell Signaling Technology 9542 AMIGO2 (for IHC) Abnova H00347902-B01P Lot# F8271 RRID:AB_1672842 AMIGO2 (for IF and WB) R&D systems AF2080 PTK7 (N-terminal) (for WB) R&D systems AF4499 PTK7 (C-terminal) (for IF and WB) Cell Signaling 11926 Secondary rabbit anti goat (for AMIGO2 detection on WB) Sigma A5420 HRP-conjugated ACTIN Sigma A3854 Biological Samples Melanoma and normal skin tissue microarray Biomax ME208 Nevus tissue microarray IMCG, NYU MT032808 Patient derived short term cultures Osman Laboratory, NYU de Miera et al., 2012 NHM (this paper) Bernstein Laboratory, ISMMS NHM1-5 Chemicals, Peptides, and Recombinant Proteins GFP Trap-A beads Chromotek gta-10 BET inhibitor JQ1 Bradner Laboratory; Agreement # {"type":"entrez-nucleotide","attrs":{"text":"A09608","term_id":"490581","term_text":"A09608"}} A09608 N/A BET inhibitor I-BET762 Apex Bio 501014747 IGEPAL Sigma I-8896 Micrococcal Nuclease Affymetrix 70196Y Dynabeads Protein A Immunoprécipitation kit Invitrogen 10006D Magna ChIP Protein A+G Magnetic beads Millipore 16-663 DSG Pierce (ThermoFisher) 20593 Critical Commercial Assays Click- Click-IT EdU Pacific Blue Flow Cytometry Assay Kit ThermoFisher {"type":"entrez-nucleotide","attrs":{"text":"C10636","term_id":"1535707","term_text":"C10636"}} C10636 Caspase Activity assay MBL Int.

Techniques:

Journal: Molecular cell

Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

doi: 10.1016/j.molcel.2017.11.004

Figure Lengend Snippet: (A) Schematic of AMIGO2 SE showing enhancers E1–E5 (B), deleted sequences for CRISPR-Cas9 editing (C), and DNA segments used for E3 lucif-erase assay (D).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies BRD2 Bethyl Laboratories A302-583A BRD4 Bethyl Laboratories A301-985A H3K4me1 Abcam ab8895 H3K4me3 Abcam ab1012 H3K27ac Abcam ab177178 H3K27me3 EMD 07-449 MED1 Bethyl Laboratories A300-793A macroH2A2 Bernstein Laboratory, ISMMS in house macroH2A1 Abcam ab37264 CTCF EMD 07-729 FOSL2 Santa Cruz SC-604 TEAD4 Santa Cruz SC-101184 HSP90 Santa Cruz SC-13119 GAPDH Santa Cruz SC-32233 GFP Roche 11814460001 LAMIN B1 Santa Cruz SC-6217 ALCAM Abcam ab109215 L1CAM Abcam ab20148 NRP2 Santa Cruz SC-13117 NRP1 Abcam ab81321 TUBULIN Sigma T9026 FOXM1 Cell Signaling Technology 5436 Casp3 Cell Signaling Technology 9662 PARP Cell Signaling Technology 9542 AMIGO2 (for IHC) Abnova H00347902-B01P Lot# F8271 RRID:AB_1672842 AMIGO2 (for IF and WB) R&D systems AF2080 PTK7 (N-terminal) (for WB) R&D systems AF4499 PTK7 (C-terminal) (for IF and WB) Cell Signaling 11926 Secondary rabbit anti goat (for AMIGO2 detection on WB) Sigma A5420 HRP-conjugated ACTIN Sigma A3854 Biological Samples Melanoma and normal skin tissue microarray Biomax ME208 Nevus tissue microarray IMCG, NYU MT032808 Patient derived short term cultures Osman Laboratory, NYU de Miera et al., 2012 NHM (this paper) Bernstein Laboratory, ISMMS NHM1-5 Chemicals, Peptides, and Recombinant Proteins GFP Trap-A beads Chromotek gta-10 BET inhibitor JQ1 Bradner Laboratory; Agreement # {"type":"entrez-nucleotide","attrs":{"text":"A09608","term_id":"490581","term_text":"A09608"}} A09608 N/A BET inhibitor I-BET762 Apex Bio 501014747 IGEPAL Sigma I-8896 Micrococcal Nuclease Affymetrix 70196Y Dynabeads Protein A Immunoprécipitation kit Invitrogen 10006D Magna ChIP Protein A+G Magnetic beads Millipore 16-663 DSG Pierce (ThermoFisher) 20593 Critical Commercial Assays Click- Click-IT EdU Pacific Blue Flow Cytometry Assay Kit ThermoFisher {"type":"entrez-nucleotide","attrs":{"text":"C10636","term_id":"1535707","term_text":"C10636"}} C10636 Caspase Activity assay MBL Int.

Techniques: CRISPR

Journal: Molecular cell

Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

doi: 10.1016/j.molcel.2017.11.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies BRD2 Bethyl Laboratories A302-583A BRD4 Bethyl Laboratories A301-985A H3K4me1 Abcam ab8895 H3K4me3 Abcam ab1012 H3K27ac Abcam ab177178 H3K27me3 EMD 07-449 MED1 Bethyl Laboratories A300-793A macroH2A2 Bernstein Laboratory, ISMMS in house macroH2A1 Abcam ab37264 CTCF EMD 07-729 FOSL2 Santa Cruz SC-604 TEAD4 Santa Cruz SC-101184 HSP90 Santa Cruz SC-13119 GAPDH Santa Cruz SC-32233 GFP Roche 11814460001 LAMIN B1 Santa Cruz SC-6217 ALCAM Abcam ab109215 L1CAM Abcam ab20148 NRP2 Santa Cruz SC-13117 NRP1 Abcam ab81321 TUBULIN Sigma T9026 FOXM1 Cell Signaling Technology 5436 Casp3 Cell Signaling Technology 9662 PARP Cell Signaling Technology 9542 AMIGO2 (for IHC) Abnova H00347902-B01P Lot# F8271 RRID:AB_1672842 AMIGO2 (for IF and WB) R&D systems AF2080 PTK7 (N-terminal) (for WB) R&D systems AF4499 PTK7 (C-terminal) (for IF and WB) Cell Signaling 11926 Secondary rabbit anti goat (for AMIGO2 detection on WB) Sigma A5420 HRP-conjugated ACTIN Sigma A3854 Biological Samples Melanoma and normal skin tissue microarray Biomax ME208 Nevus tissue microarray IMCG, NYU MT032808 Patient derived short term cultures Osman Laboratory, NYU de Miera et al., 2012 NHM (this paper) Bernstein Laboratory, ISMMS NHM1-5 Chemicals, Peptides, and Recombinant Proteins GFP Trap-A beads Chromotek gta-10 BET inhibitor JQ1 Bradner Laboratory; Agreement # {"type":"entrez-nucleotide","attrs":{"text":"A09608","term_id":"490581","term_text":"A09608"}} A09608 N/A BET inhibitor I-BET762 Apex Bio 501014747 IGEPAL Sigma I-8896 Micrococcal Nuclease Affymetrix 70196Y Dynabeads Protein A Immunoprécipitation kit Invitrogen 10006D Magna ChIP Protein A+G Magnetic beads Millipore 16-663 DSG Pierce (ThermoFisher) 20593 Critical Commercial Assays Click- Click-IT EdU Pacific Blue Flow Cytometry Assay Kit ThermoFisher {"type":"entrez-nucleotide","attrs":{"text":"C10636","term_id":"1535707","term_text":"C10636"}} C10636 Caspase Activity assay MBL Int.

Techniques: Microarray, Derivative Assay, Recombinant, Magnetic Beads, Flow Cytometry, Caspase Activity Assay, Blocking Assay, TA Cloning, Sequencing, RNA Sequencing Assay, Western Blot, Mass Spectrometry, Expressing, Software

Journal: International Journal of Molecular Sciences

Article Title: Tamoxifen Application Is Associated with Transiently Increased Loss of Hippocampal Neurons following Virus Infection

doi: 10.3390/ijms22168486

Figure Lengend Snippet: Immunohistochemical evaluation of hippocampi of Theiler’s murine encephalomyelitis virus BeAn (TMEV-BeAn) infected mice 14 days post-infection (dpi). Further differentiation of the astrocyte phenotypes into amigo 2-positive cytotoxic A1 astrocytes and S100A10-positive neurotrophic A2 astrocytes revealed an increase of both phenotypes in tamoxifen-treated animals. The number of cytotoxic A1 astrocytes was higher in tamoxifen-treated animals compared to untreated animals with hippocampal neuronal loss. Aquaporin 4 expression was downregulated in the presence of inflammation but was not further impacted by tamoxifen application. Scale bars: 100 µm (Amigo 2, S100A10, Aquaporin 4). Statistical significant differences are depicted as * = p ≤ 0.05 ( for each p -value individually).

Article Snippet: The following primary antibodies were used as previously described [ ]: polyclonal rabbit-anti-glial fibrillary acidic protein (GFAP) antibody (Agilent Technologies, Inc., Santa Clara, CA, USA, Dako, cat. Z0334, 1:1000), in-house polyclonal rabbit-anti-TMEV antibody (1:2000), monoclonal mouse-anti-neuron nuclear antigen (NeuN) antibody (Merck-Millipore, Inc., Burlington, MA, USA, cat. MAB377, MW, 1:1600), monoclonal mouse-anti-doublecortin (DCX) antibody (Santa-Cruz Biotechnology, Inc., Dallas, TX, USA, cat. Sc-271390, 1:100), polyclonal rabbit-anti-nestin antibody (OriGene Technologies, Inc., Rockville, MD, USA, cat. AP07829PU-N, 1:500), polyclonal rabbit-anti-ionized calcium-binding adapter molecule-1 (IBA-1) antibody (Invitrogen, Thermo Firscher Scientific, Waltham, MA, USA, cat. PA5-27436, 1:2000), polyclonal rabbit-anti-human CD3 (Agilent Technologies, Inc., Santa Clara, CA, USA, Dako, cat. A0452, MW, 1:250), anti-adhesion molecule with Ig-like domain 2–Type 1 astroglial (amigo 2) antibody (Bioss, Inc., Woburn, MA, USA, cat. Bs-11450R, T, 1:200), anti-S100A10—Type 2 astroglial antibody (Bio-Techne GmbH, Wiesbaden, Germany, cat. Ab JF0987, 1:100), anti-aquaporin 4 (Merck-Millipore, Inc., Burlington, MA, USA cat. AB3594, 1:200).

Techniques: Immunohistochemical staining, Infection, Expressing

Journal: The Journal of Cell Biology

Article Title: AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation

doi: 10.1083/jcb.201503113

Figure Lengend 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.

Article Snippet: Full-length human AMIGO2 was purchased from GeneCopoeia (EX-E1271-M02) and subcloned into the EGFP-tagged vector using HindIII (5′-CCCAAGCTTGGCGACCATAATGTCGTTACGTGTACACACT-3′) and BamHI (5′-CGGGATCCCGTTAAGTGGACGCCACAAAAG-3′).

Techniques: Transfection, Immunostaining, Two Tailed Test, Expressing, Flow Cytometry, Staining, Migration, Control

Journal: The Journal of Cell Biology

Article Title: AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation

doi: 10.1083/jcb.201503113

Figure Lengend 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.

Article Snippet: Full-length human AMIGO2 was purchased from GeneCopoeia (EX-E1271-M02) and subcloned into the EGFP-tagged vector using HindIII (5′-CCCAAGCTTGGCGACCATAATGTCGTTACGTGTACACACT-3′) and BamHI (5′-CGGGATCCCGTTAAGTGGACGCCACAAAAG-3′).

Techniques: In Vivo, Injection, Control, shRNA, Staining, TUNEL Assay, Immunostaining, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation

doi: 10.1083/jcb.201503113

Figure Lengend 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.

Article Snippet: Full-length human AMIGO2 was purchased from GeneCopoeia (EX-E1271-M02) and subcloned into the EGFP-tagged vector using HindIII (5′-CCCAAGCTTGGCGACCATAATGTCGTTACGTGTACACACT-3′) and BamHI (5′-CGGGATCCCGTTAAGTGGACGCCACAAAAG-3′).

Techniques: Knockdown, Over Expression, Activation Assay, Phospho-proteomics, Protein-Protein interactions, Transfection

Journal: The Journal of Cell Biology

Article Title: AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation

doi: 10.1083/jcb.201503113

Figure Lengend 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.

Article Snippet: Full-length human AMIGO2 was purchased from GeneCopoeia (EX-E1271-M02) and subcloned into the EGFP-tagged vector using HindIII (5′-CCCAAGCTTGGCGACCATAATGTCGTTACGTGTACACACT-3′) and BamHI (5′-CGGGATCCCGTTAAGTGGACGCCACAAAAG-3′).

Techniques: Immunostaining, Membrane, Cell Fractionation, Western Blot, Control, Immunoprecipitation, Clinical Proteomics

Journal: The Journal of Cell Biology

Article Title: AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation

doi: 10.1083/jcb.201503113

Figure Lengend 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.

Article Snippet: Full-length human AMIGO2 was purchased from GeneCopoeia (EX-E1271-M02) and subcloned into the EGFP-tagged vector using HindIII (5′-CCCAAGCTTGGCGACCATAATGTCGTTACGTGTACACACT-3′) and BamHI (5′-CGGGATCCCGTTAAGTGGACGCCACAAAAG-3′).

Techniques: Transfection, Immunoprecipitation, Construct, Incubation, Purification, Western Blot, Control, Sequencing, Mutagenesis, Staining

Journal: The Journal of Cell Biology

Article Title: AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation

doi: 10.1083/jcb.201503113

Figure Lengend 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.

Article Snippet: Full-length human AMIGO2 was purchased from GeneCopoeia (EX-E1271-M02) and subcloned into the EGFP-tagged vector using HindIII (5′-CCCAAGCTTGGCGACCATAATGTCGTTACGTGTACACACT-3′) and BamHI (5′-CGGGATCCCGTTAAGTGGACGCCACAAAAG-3′).

Techniques: Translocation Assay, Activation Assay, Binding Assay, Incubation, Fluorescence, Two Tailed Test, Competitive Binding Assay, Purification, Western Blot, Immunoprecipitation, Clinical Proteomics, Membrane, Cell Fractionation, Control

Journal: The Journal of Cell Biology

Article Title: AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation

doi: 10.1083/jcb.201503113

Figure Lengend 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.

Article Snippet: Full-length human AMIGO2 was purchased from GeneCopoeia (EX-E1271-M02) and subcloned into the EGFP-tagged vector using HindIII (5′-CCCAAGCTTGGCGACCATAATGTCGTTACGTGTACACACT-3′) and BamHI (5′-CGGGATCCCGTTAAGTGGACGCCACAAAAG-3′).

Techniques: Comparison, TUNEL Assay, shRNA, Transfection

Journal: The Journal of Cell Biology

Article Title: AMIGO2, a novel membrane anchor of PDK1, controls cell survival and angiogenesis via Akt activation

doi: 10.1083/jcb.201503113

Figure Lengend 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.

Article Snippet: Full-length human AMIGO2 was purchased from GeneCopoeia (EX-E1271-M02) and subcloned into the EGFP-tagged vector using HindIII (5′-CCCAAGCTTGGCGACCATAATGTCGTTACGTGTACACACT-3′) and BamHI (5′-CGGGATCCCGTTAAGTGGACGCCACAAAAG-3′).

Techniques: Binding Assay, Activation Assay, Clinical Proteomics, Membrane, Migration, Phospho-proteomics