Journal: bioRxiv

Article Title: The AMIGO1 adhesion protein activates Kv2.1 voltage sensors

doi: 10.1101/2021.06.20.448455

Figure Lengend Snippet: ( A ) Experimental set up: Whole-cell K + currents (arrow) from Kv2.1–CHO transfected with GFP (red), rAMIGO2–YFP (dark blue), or rAMIGO3–YFP (light blue). Same voltage protocols and representation as . ( B , C, D ) Representative Kv2.1–control (5.1 pF), Kv2.1 + AMIGO2 (6.6 pF) or Kv2.1 + AMIGO3 (2.4 pF) cells. ( E , F, G ) Normalized G–V relationships. 5 of the Kv2.1–control cells were recorded from side by side with the Kv2.1 + AMIGO2 cells and Kv2.1 + AMIGO3 cells (light red). There was no statistical difference between these 5 cells and the data previously acquired during Kv2.1 + AMIGO1 recordings for (assessed by t-test), and data was pooled. Solid lines a 4 th order Boltzmann fits . ( H ) Reconstructed 4 th order Boltzmann fits from average V i,Mid and z i . Shading V i,Mid ± SEM. ( I ) Steepness and ( J ) midpoint of fits. Statistics in . ***: p = ≤0.001, **: p = ≤0.01, *: p = ≤0.05. Bars are mean ± SEM.

Article Snippet: Cells were incubated in the transfection cocktail and 2 mL of unsupplemented media for 6-8 hours before being returned to regular growth media, and used for experiments 40-48 hours after transfection. pEGFP, mAMIGO1–pEYFP–N1, and pCAG–ChroME-mRuby2-ST ( ) plasmids were gifts from James Trimmer. mAMIGO1–pEYFP–N1 uses a VPRARDPPVAT linker to tag the internal C–terminus of wild–type mouse AMIGO1 (NM_001004293.2 or NM_146137.3) with eYFP. pCAG– ChroME–mRuby2–ST encodes an mRuby2–tagged channelrhodopsin with a Kv2.1 PRC trafficking sequence ( , ). mKv2.1 (NM_008420) was purchased from OriGene (MG210968). hSCN1β–pIRES2–GFP was a gift from Vladimir Yarov-Yarovoy. mAMIGO1 was subcloned into pIRES2–GFP between NheI and BamHI restriction sites. rAMIGO2–pEYFP–N1 and rAMIGO3–pEYFP–N1 were generated by subcloning rat AMIGO2 (NM_182816.2) or rat AMIGO3 (NM_178144.1) in place of mAMIGO1 in the mAMIGO1–pEYFP–N1 vector.

Techniques: Transfection

Journal: bioRxiv

Article Title: The AMIGO1 adhesion protein activates Kv2.1 voltage sensors

doi: 10.1101/2021.06.20.448455

Figure Lengend Snippet: ( A ) Coefficient of variation of fluorescence from AMIGO2–YFP (dark blue circles), AMIGO3–YFP (light blue circles), or GxTX–594 (red circles). COV from confocal images of glass-adhered membranes (exemplar images in B-G ). AMIGO2-YFP fluorescence from cells ( B ) not induced for Kv2.1 expression (COVΔ2,oh = 0.2090 ± 0.0062, n = 144), ( C ) induced 48 h for Kv2.1 expression (COV A2,48h = 0.342 ± 0.022, n = 65). ( D ) GxTX–594 labeling of the cells in C (COV A2,48h(GxTX–594) = 0.631 ± 0.013, n = 65 cells). AMIGO3–YFP fluorescence from cells ( E ) not induced for Kv2.1 expression (COV A3,0h = 0.2186 ± 0.0052, n = 160), ( F ) induced 48 h for Kv2.1 expression (COV A3,48h = 0.503 ± 0.014, n = 109). ( G ) GxTX–594 labeling of the cells in panel F (COV A3,48h(GxTX–594) = 0.650 ± 0.013, n = 109 cells). ( H ) Costes thresholded, Pearson’s colocalization coefficients from cells induced for Kv2.1 expression 48 h prior to imaging.. From left to right: PCC A2,GxTX–594 = 0.342 ± 0.022, ≥ 0 (p < 0.0001, one–tailed, t-test), n = 65; PCC 594 = 0.597 ± 0.020, ≥ 0 (p < 0.0001, one–tailed, t-test), n = 108. ( I, J, K) Exemplar images where merge overlay (white) shows colocalization between GxTX–594 (red) and AMIGO2-YFP (cyan) or (L, M, N ) AMIGO2-YFP (cyan) Arithmetic means and standard errors are plotted. ( Statistics ) Outliers were removed using ROUT, Q = 1%. An ordinary one-way ANOVA with multiple comparisons was used to evaluate the differences between groups in COV analysis, while a t-test was used to evaluate the PCC data. ****: p = ≤0.0001. Bars are mean ± SEM. All scale bars are 10 μm.

Article Snippet: Cells were incubated in the transfection cocktail and 2 mL of unsupplemented media for 6-8 hours before being returned to regular growth media, and used for experiments 40-48 hours after transfection. pEGFP, mAMIGO1–pEYFP–N1, and pCAG–ChroME-mRuby2-ST ( ) plasmids were gifts from James Trimmer. mAMIGO1–pEYFP–N1 uses a VPRARDPPVAT linker to tag the internal C–terminus of wild–type mouse AMIGO1 (NM_001004293.2 or NM_146137.3) with eYFP. pCAG– ChroME–mRuby2–ST encodes an mRuby2–tagged channelrhodopsin with a Kv2.1 PRC trafficking sequence ( , ). mKv2.1 (NM_008420) was purchased from OriGene (MG210968). hSCN1β–pIRES2–GFP was a gift from Vladimir Yarov-Yarovoy. mAMIGO1 was subcloned into pIRES2–GFP between NheI and BamHI restriction sites. rAMIGO2–pEYFP–N1 and rAMIGO3–pEYFP–N1 were generated by subcloning rat AMIGO2 (NM_182816.2) or rat AMIGO3 (NM_178144.1) in place of mAMIGO1 in the mAMIGO1–pEYFP–N1 vector.

Techniques: Fluorescence, Expressing, Labeling, Imaging, One-tailed Test

Journal: Diagnostic Pathology

Article Title: Establishment of an antibody specific for AMIGO2 improves immunohistochemical evaluation of liver metastases and clinical outcomes in patients with colorectal cancer

doi: 10.1186/s13000-021-01176-2

Figure Lengend Snippet: Specific detection of AMIGO2 by the monoclonal antibody rTNK1A0012. The large membrane was reacted with anti-AMIGO2 antibody, while the small membrane was reacted with an anti-β-actin antibody. A Cell lysates were prepared from HepG2 cells transfected with human AMIGO1 (A1), AMIGO2 (A2), AMIGO3 (A3), and an empty vector (E). Immunoblotting with rTNK1A0012 (rTNK mAb). B Immunoblotting with sc-373699 (sc mAb). C Cell lysates were treated with or without PNGase F and immunoblotted with rTNK mAb. D Cell lysates were treated with or without alkaline β-elimination and immunoblotted with rTNK mAb

Article Snippet: Expression plasmids for human AMIGO1 (pEZ-M02/AMIGO1), AMIGO2 (pEZ-M02/AMIGO2), and AMIGO3 (pEZ-M02/AMIGO3) were purchased from GeneCopoeia (EX-E1371-M02, EX-E1271-M02, EX-E1133-M02, Rockville, MD).

Techniques: Membrane, Transfection, Plasmid Preparation, Western Blot

Journal: Diagnostic Pathology

Article Title: Establishment of an antibody specific for AMIGO2 improves immunohistochemical evaluation of liver metastases and clinical outcomes in patients with colorectal cancer

doi: 10.1186/s13000-021-01176-2

Figure Lengend Snippet: Detection of three types of AMIGO family molecules by commercially available antibodies. A The same cell lysates as shown in Fig. was used. Lysates were prepared from HepG2 cells transfected with human AMIGO1 (A1), AMIGO2 (A2), AMIGO3 (A3), and an empty vector (E). Immunoblotting with LS-C404504 polyclonal antibody. B Immunoblotting with #36094 polyclonal antibody. C Immunoblotting with HPA054004 polyclonal antibody

Article Snippet: Expression plasmids for human AMIGO1 (pEZ-M02/AMIGO1), AMIGO2 (pEZ-M02/AMIGO2), and AMIGO3 (pEZ-M02/AMIGO3) were purchased from GeneCopoeia (EX-E1371-M02, EX-E1271-M02, EX-E1133-M02, Rockville, MD).

Techniques: Transfection, Plasmid Preparation, Western Blot

Journal: Diagnostic Pathology

Article Title: Establishment of an antibody specific for AMIGO2 improves immunohistochemical evaluation of liver metastases and clinical outcomes in patients with colorectal cancer

doi: 10.1186/s13000-021-01176-2

Figure Lengend Snippet: Immunohistochemical staining for AMIGO2 expression by the monoclonal antibody rTNK1A0012 in CRC tissues. AMIGO2 negative expression ( A and C ) and AMIGO2 high expression ( B and D ) are shown. The lower row are shown at higher magnification. Scale bars = 400 μm ( A and B ) and 100 μm ( C and D )

Article Snippet: Expression plasmids for human AMIGO1 (pEZ-M02/AMIGO1), AMIGO2 (pEZ-M02/AMIGO2), and AMIGO3 (pEZ-M02/AMIGO3) were purchased from GeneCopoeia (EX-E1371-M02, EX-E1271-M02, EX-E1133-M02, Rockville, MD).

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Diagnostic Pathology

Article Title: Establishment of an antibody specific for AMIGO2 improves immunohistochemical evaluation of liver metastases and clinical outcomes in patients with colorectal cancer

doi: 10.1186/s13000-021-01176-2

Figure Lengend Snippet: AMIGO2 expression and clinicopathological factors affecting overall survival rate in 173 CRC patients

Article Snippet: Expression plasmids for human AMIGO1 (pEZ-M02/AMIGO1), AMIGO2 (pEZ-M02/AMIGO2), and AMIGO3 (pEZ-M02/AMIGO3) were purchased from GeneCopoeia (EX-E1371-M02, EX-E1271-M02, EX-E1133-M02, Rockville, MD).

Techniques: Expressing

Journal: Diagnostic Pathology

Article Title: Establishment of an antibody specific for AMIGO2 improves immunohistochemical evaluation of liver metastases and clinical outcomes in patients with colorectal cancer

doi: 10.1186/s13000-021-01176-2

Figure Lengend Snippet: Relationship between AMIGO2 expression in primary colorectal cancer and metastatic site. A high expression of AMIGO2 was significantly associated with liver metastases ( A ) and lung metastases ( B ), but not with peritoneal dissemination ( C ), as calculated using the X 2 test

Article Snippet: Expression plasmids for human AMIGO1 (pEZ-M02/AMIGO1), AMIGO2 (pEZ-M02/AMIGO2), and AMIGO3 (pEZ-M02/AMIGO3) were purchased from GeneCopoeia (EX-E1371-M02, EX-E1271-M02, EX-E1133-M02, Rockville, MD).

Techniques: Expressing

Journal: Diagnostic Pathology

Article Title: Establishment of an antibody specific for AMIGO2 improves immunohistochemical evaluation of liver metastases and clinical outcomes in patients with colorectal cancer

doi: 10.1186/s13000-021-01176-2

Figure Lengend Snippet: Univariate and multivariate analysis for overall survival in CRC patients (Cox proportional hazard regression model)

Article Snippet: Expression plasmids for human AMIGO1 (pEZ-M02/AMIGO1), AMIGO2 (pEZ-M02/AMIGO2), and AMIGO3 (pEZ-M02/AMIGO3) were purchased from GeneCopoeia (EX-E1371-M02, EX-E1271-M02, EX-E1133-M02, Rockville, MD).

Techniques: Expressing

Journal: Diagnostic Pathology

Article Title: Establishment of an antibody specific for AMIGO2 improves immunohistochemical evaluation of liver metastases and clinical outcomes in patients with colorectal cancer

doi: 10.1186/s13000-021-01176-2

Figure Lengend Snippet: Univariate and multivariate analysis of factors affecting liver metastases in CRC patients (Logistic regression model)

Article Snippet: Expression plasmids for human AMIGO1 (pEZ-M02/AMIGO1), AMIGO2 (pEZ-M02/AMIGO2), and AMIGO3 (pEZ-M02/AMIGO3) were purchased from GeneCopoeia (EX-E1371-M02, EX-E1271-M02, EX-E1133-M02, Rockville, MD).

Techniques: Expressing

Journal: Diagnostic Pathology

Article Title: Establishment of an antibody specific for AMIGO2 improves immunohistochemical evaluation of liver metastases and clinical outcomes in patients with colorectal cancer

doi: 10.1186/s13000-021-01176-2

Figure Lengend Snippet: Comparison of two AMIGO2 antibodies in AMIGO2 expression and survival in CRC patients. Cumulative survival rates were assessed using the Kaplan-Meier plot method. Differences were analyzed using the log-rank test. Overall survival ( A ) and disease-specific survival ( B ) are shown

Article Snippet: Expression plasmids for human AMIGO1 (pEZ-M02/AMIGO1), AMIGO2 (pEZ-M02/AMIGO2), and AMIGO3 (pEZ-M02/AMIGO3) were purchased from GeneCopoeia (EX-E1371-M02, EX-E1271-M02, EX-E1133-M02, Rockville, MD).

Techniques: Comparison, Expressing