Journal: STAR Protocols

Article Title: Protocol for 3D-guided sectioning and deep cell phenotyping via light sheet imaging and 2D spatial multiplexing

doi: 10.1016/j.xpro.2025.104296

Figure Lengend Snippet: 3D light sheet and 2D multi-cyclic imaging data comparison (Mouse Glioblastoma) (A) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red). (B) Imaris 3D surface rendering of autofluorescence (cyan) and glioblastoma target cells stained with anti-GFP-Alexa Fluor 647 nanobody (red) with target plane in yellow. (C) Optical section of target plane of interest. (D) Fluorescence image of physical cryosection. (E) MICS image of section shown in D. (F) MICS image indicating anti-GFP-Alexa Fluor 647 nanobody (red) staining. (G) Magnified merged four color multiparameter MICS image with anti-EGFR (magenta), anti-GFAP (green), anti-NeuN (blue), anti-CD146 (yellow). (H–P) Nine exemplary MICS images with merges of anti-GFP-Alexa Fluor 647 nanobody staining (red) and antibody-conjugates against EGFR (H), Neurofilament (I), Nestin (J), GFAP (K), CD44 (L), CD146 (M), NeuN (N), EphA2 (O) and GLAST (P) (gray) (see “Antibodies”). Scale bars: (A–F) 500 μm; (G) 50 μm; (H–P) 500 μm.

Article Snippet: EphA2 antibody, anti-mouse, APC, REAfinity , Miltenyi Biotec B.V. & Co. KG , Cat# 130-109-187 RRID: AB_2651638.

Techniques: Imaging, Comparison, Staining, Fluorescence

Journal: Clinical Cancer Research

Article Title: Expression of EphA2 and Ephrin A-1 in Carcinoma of the Urinary Bladder

doi: 10.1158/1078-0432.ccr-05-1505

Figure Lengend Snippet: Fig. 1. A, EphA2 expression in human urinary bladder cancer cells.Twenty-five micrograms of cell proteins were resolved by SDS-PAGE.Western blot analysis was done using EphA2-specific antibody (D7) and detected using chemiluminescence. h-Actin antibody confirmed equalloading of proteins.The molecular weight markers are indicated on the left. B, analysis of theWestern blot by densitometry.The EphA2 proteinlevel is indicated as the ratio of EphA2 protein to h-actin protein. C, real-time PCR analysis of EphA2 mRNA.Total RNA isolated from bladder cancer cell lines indicated were analyzed by real-time PCR as described in Materials and Methods. Relative EphA2 mRNA levels and ratio of EphA2 mRNA/GAPDH mRNA. Bars, SD.

Article Snippet: Specificity of EphA2 and Ephrin A-1 antibodies was detected by competition with EphA2 protein or with the peptide (Santa Cruz Biotechnology) used to raise Ephrin A-1 antibody, respectively.

Techniques: Expressing, SDS Page, Western Blot, Molecular Weight, Real-time Polymerase Chain Reaction, Isolation

Journal: Clinical Cancer Research

Article Title: Expression of EphA2 and Ephrin A-1 in Carcinoma of the Urinary Bladder

doi: 10.1158/1078-0432.ccr-05-1505

Figure Lengend Snippet: Fig. 2. EphA2, Ephrin A-1, and E-cadherin expression in urothelial tissues. Expression of EphA2 (A-C), Ephrin A-1 (D-F), and E-cadherin (G-I) in normal urothelium (A, D, and G),Ta stage (B, E, and H), andT4 stage (C, E, and F) tumors.Very low intensity staining is observed for EphA2 in the normal urothelium (A) whereas very intense stainingis observedinthe cytoplasm of tumor cells (B and C). Basal expressionof Ephrin A-1is observed in normal urothelium (D) whereas very high staining pattern is observed in the cytoplasm of tumor cells (E and F).Very intense staining of the cytomembrane of the normal urothelial cells is observed for E-cadherin (G) whereas less frequent cytoplasmic staining and low intensity staining of the membrane are observed in bladder cancer cells (H and I). Magnification, 20.

Article Snippet: Specificity of EphA2 and Ephrin A-1 antibodies was detected by competition with EphA2 protein or with the peptide (Santa Cruz Biotechnology) used to raise Ephrin A-1 antibody, respectively.

Techniques: Expressing, Staining, Membrane

Journal: Clinical Cancer Research

Article Title: Expression of EphA2 and Ephrin A-1 in Carcinoma of the Urinary Bladder

doi: 10.1158/1078-0432.ccr-05-1505

Figure Lengend Snippet: Fig. 4. A, E-cadherin expression in bladder cancer cell lines.Twenty-five micrograms of protein from whole-celllysates were fractionated on 7.5% SDS-PAGE gel.The blot was probed with E-cadherin antibody, then stripped and reprobed with h-actin antibody for loading control. B, EphA2 phosphorylation in bladder cancer cells. EphA2 protein was immunoprecipitated from100 Ag of whole-cell lysates and resolved by SDS-PAGE.The blots were probed with p-Tyr antibody-4G10 (top) and detected by chemiluminescence. Duplicate cell lysates were resolved simultaneously on the same gel and probed with EphA2-specific antibody (D7; bottom). EphA2 proteinlevel from each cell lysate is indicated.TCCSUP treated with Ephrin A-1-Fc was the positive control and sepharose beads linked to immunoglobulin G were the negative control. Molecular weight markers are indicated on the left.

Article Snippet: Specificity of EphA2 and Ephrin A-1 antibodies was detected by competition with EphA2 protein or with the peptide (Santa Cruz Biotechnology) used to raise Ephrin A-1 antibody, respectively.

Techniques: Expressing, SDS Page, Control, Phospho-proteomics, Immunoprecipitation, Positive Control, Negative Control, Molecular Weight

Journal: Clinical Cancer Research

Article Title: Expression of EphA2 and Ephrin A-1 in Carcinoma of the Urinary Bladder

doi: 10.1158/1078-0432.ccr-05-1505

Figure Lengend Snippet: Fig. 5. A, degradation of EphA2 protein with Ephrin A-1stimulation.TCCSUP cells were incubated in the presence of1 Ag/mL Ephrin A-1-Fc at 37jC for 0 minutes to 4 hours.Twenty-five micrograms of protein from each cell lysates were fractionated on SDS-PAGE.Western blot analysis was carried out with the EphA2-specific antibody D7 and detectedusing chemiluminescence. h-Catenin antibody confirmed equal sample loading. Molecular weight markers are indicated on the left. B, degradation of EphA2 occurs via phosphorylation.TCCSUP cells were incubated in the presence of1 Ag/mL Ephrin A-1-Fc at 37jC for 0 minutes to 4 hours. Immunoprecipitates of EphA2 from cell lysates were fractionated on SDS-PAGE. Western blot analysis was done using p-Tyr antibody (4G10; A) and detected using chemiluminescence kit. Duplicate cell lysates were resolved simultaneously on the same gel and probed with EphA2-specific antibody (D7; B). Sepharose beads linked to immunoglobulin G were included as negative control.

Article Snippet: Specificity of EphA2 and Ephrin A-1 antibodies was detected by competition with EphA2 protein or with the peptide (Santa Cruz Biotechnology) used to raise Ephrin A-1 antibody, respectively.

Techniques: Incubation, SDS Page, Western Blot, Molecular Weight, Phospho-proteomics, Negative Control

Journal: Journal of Virology

Article Title: A Kaposi's Sarcoma-Associated Herpesvirus Infection Mechanism Is Independent of Integrins α3β1, αVβ3, and αVβ5

doi: 10.1128/JVI.00803-18

Figure Lengend Snippet: CRISPR-Cas9 guide RNA sequences used to target the indicated genes

Article Snippet: Heparan sulfate antibody (F58-10E4) was purchased from Amsbio; integrin α3 antibody (P1B5) was purchased from Calbiochem; integrin αV, integrin β7, EphA2, and EphA5 antibodies (MAB12191, MAB4669, AF3035, and MAB541, respectively) were obtained from R&D Systems; integrin β1 and integrin β3 antibodies (T2S/16 and PM6/13, respectively) were obtained from Novus Biologicals; integrin β5 and EphA2 antibodies (AST-3T and SHM16, respectively) were obtained from BioLegend; xCT and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies (ab37185 and ab181602, respectively) were obtained from Abcam; DC-SIGN antibody (DCN47.5) was obtained from Miltenyi Biotec; EphA4 antibody (4C8H5) was obtained from ThermoFisher; EphB2 antibody (2D12C6) was obtained from Santa Cruz Biotech; and Flag antibody (M2) was obtained from Sigma-Aldrich.

Techniques: CRISPR, Sequencing

Journal: Journal of Virology

Article Title: A Kaposi's Sarcoma-Associated Herpesvirus Infection Mechanism Is Independent of Integrins α3β1, αVβ3, and αVβ5

doi: 10.1128/JVI.00803-18

Figure Lengend Snippet: EphA4 and EphB2 are dispensable for infection of Caki-1 cells. (A, E, F, and H) A total of 120 μg (A, E, and H) or 50 μg (F) of the indicated whole-cell lysate proteins was run on a 10% SDS-PAGE gel and blotted for EphA4 (A and E) or EphB2 (F and H) and GAPDH as a loading control. (B and C) WT and EPHA4 KO Caki-1 cells (B) or WT, EPHA2 KO, and EPHA4/EPHA2 DKO Caki-1 cells (C) were infected with KSHV in triplicate, and the infection percentage was measured by flow cytometry at 2 days postinfection. The infection percentages of KO cell lines were normalized to the average infection percentage of WT cells, and data from a representative experiment are shown. (D) EPHA4 KO and EPHA4/EPHA2 DKO Caki-1 cells were immunostained for surface EphA2 expression. Gray histograms represent isotype controls. (G) WT and EPHB2 KO Caki-1 cells were infected with KSHV in triplicate, and infection percentages were quantified by flow cytometry at 2 days postinfection. The rates of infection of the KO line were normalized to the average WT infection rate, and data from a representative experiment are shown. *, P < 0.05.

Article Snippet: Heparan sulfate antibody (F58-10E4) was purchased from Amsbio; integrin α3 antibody (P1B5) was purchased from Calbiochem; integrin αV, integrin β7, EphA2, and EphA5 antibodies (MAB12191, MAB4669, AF3035, and MAB541, respectively) were obtained from R&D Systems; integrin β1 and integrin β3 antibodies (T2S/16 and PM6/13, respectively) were obtained from Novus Biologicals; integrin β5 and EphA2 antibodies (AST-3T and SHM16, respectively) were obtained from BioLegend; xCT and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies (ab37185 and ab181602, respectively) were obtained from Abcam; DC-SIGN antibody (DCN47.5) was obtained from Miltenyi Biotec; EphA4 antibody (4C8H5) was obtained from ThermoFisher; EphB2 antibody (2D12C6) was obtained from Santa Cruz Biotech; and Flag antibody (M2) was obtained from Sigma-Aldrich.

Techniques: Infection, SDS Page, Control, Flow Cytometry, Expressing

Journal: Journal of Virology

Article Title: A Kaposi's Sarcoma-Associated Herpesvirus Infection Mechanism Is Independent of Integrins α3β1, αVβ3, and αVβ5

doi: 10.1128/JVI.00803-18

Figure Lengend Snippet: Perturbations in KSHV receptor expression do not unexpectedly affect other known receptors. WT, EPHA2 KO, ITGB1 KO, and ITGAV/ITGA3 DKO Caki-1 cells were concurrently immunostained for surface expression of nontargeted, known KSHV receptors and analyzed by flow cytometry. Gray histograms represent isotype controls.

Article Snippet: Heparan sulfate antibody (F58-10E4) was purchased from Amsbio; integrin α3 antibody (P1B5) was purchased from Calbiochem; integrin αV, integrin β7, EphA2, and EphA5 antibodies (MAB12191, MAB4669, AF3035, and MAB541, respectively) were obtained from R&D Systems; integrin β1 and integrin β3 antibodies (T2S/16 and PM6/13, respectively) were obtained from Novus Biologicals; integrin β5 and EphA2 antibodies (AST-3T and SHM16, respectively) were obtained from BioLegend; xCT and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies (ab37185 and ab181602, respectively) were obtained from Abcam; DC-SIGN antibody (DCN47.5) was obtained from Miltenyi Biotec; EphA4 antibody (4C8H5) was obtained from ThermoFisher; EphB2 antibody (2D12C6) was obtained from Santa Cruz Biotech; and Flag antibody (M2) was obtained from Sigma-Aldrich.

Techniques: Expressing, Flow Cytometry

Journal: Journal of Virology

Article Title: A Kaposi's Sarcoma-Associated Herpesvirus Infection Mechanism Is Independent of Integrins α3β1, αVβ3, and αVβ5

doi: 10.1128/JVI.00803-18

Figure Lengend Snippet: EphA2 is required for infection of Caki-1 and HeLa cells. (A) WT and EPHA2 KO Caki-1 cells were immunostained for surface EphA2 expression with the SHM16 antibody and analyzed by flow cytometry. The gray histogram represents the isotype control. (B) WT and EPHA2 KO Caki-1 cells were infected with KSHV in duplicate, and infection rates were quantified by flow cytometry. The rate of infection of the EPHA2 KO pool was normalized to the average WT infection rate, and data were pooled from multiple experiments. (C) WT Caki-1 and EPHA2 KO clone A1 cells were immunostained for surface EphA2 with the antibody AF3035 and analyzed by flow cytometry. The gray histogram represents the isotype control. (D) Fifteen micrograms of whole-cell lysate protein from WT Caki-1 cells and EPHA2 KO clone A1 was run on an SDS-PAGE gel and blotted for EphA2 with AF3035 and GAPDH. (E) EPHA2 KO clone A1 Caki-1 cells were preblocked with EGFR-Fc or ephrin-A4–Fc at 10 μg/ml at 4°C and then infected in triplicate in the presence of EGFR-Fc or ephrin-A4–Fc at 5 μg/ml at 37°C. The infection percentage was measured by flow cytometry at 2 days postinfection, and percent infection was normalized to the average EPHA2 KO infection rate. (D) Mixed EPHA2 KO HeLa cells were immunostained for surface EphA2 expression with the SHM16 antibody and analyzed by flow cytometry. The gray histogram represents the isotype control. (E) Mixed EPHA2 KO HeLa cells were infected with KSHV in triplicate, and the infection percentage was measured by flow cytometry at 2 days postinfection. The cells were also immunostained for surface EphA2 and gated on EphA2-high or -low cells, as indicated in panel D. The rates of infection of EphA2-low cells were normalized to those of EphA2-high cells in each well, and data from a representative experiment are shown. *, P < 0.05.

Article Snippet: Heparan sulfate antibody (F58-10E4) was purchased from Amsbio; integrin α3 antibody (P1B5) was purchased from Calbiochem; integrin αV, integrin β7, EphA2, and EphA5 antibodies (MAB12191, MAB4669, AF3035, and MAB541, respectively) were obtained from R&D Systems; integrin β1 and integrin β3 antibodies (T2S/16 and PM6/13, respectively) were obtained from Novus Biologicals; integrin β5 and EphA2 antibodies (AST-3T and SHM16, respectively) were obtained from BioLegend; xCT and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies (ab37185 and ab181602, respectively) were obtained from Abcam; DC-SIGN antibody (DCN47.5) was obtained from Miltenyi Biotec; EphA4 antibody (4C8H5) was obtained from ThermoFisher; EphB2 antibody (2D12C6) was obtained from Santa Cruz Biotech; and Flag antibody (M2) was obtained from Sigma-Aldrich.

Techniques: Infection, Expressing, Flow Cytometry, Control, SDS Page

Journal: Journal of Virology

Article Title: A Kaposi's Sarcoma-Associated Herpesvirus Infection Mechanism Is Independent of Integrins α3β1, αVβ3, and αVβ5

doi: 10.1128/JVI.00803-18

Figure Lengend Snippet: EphA2, EphA4, and EphA5 rescue KSHV infection in EPHA2 KO Caki-1 cells. (A) Diagram of generalized full-length and PPT-3×Flag-mature ephrin receptor constructs. SAM, sterile alpha motif. (B and C) Live (B) or fixed and permeabilized (C) 3×Flag-tagged ephrin receptor-transduced EPHA2 KO cells and a vector control were immunostained for surface (B) or intracellular (C) 3×Flag expression and analyzed by flow cytometry. Gray histograms represent isotype controls. (D) The indicated cell lysates were run on 10% SDS-PAGE gels and blotted for 3×Flag, EphA4, and EphA5 with matched GAPDH as a loading control. For the Flag and EphA5 blots, 15 μg of whole-cell lysate protein was loaded. For the EphA4 blot, 120 μg of whole-cell lysate protein was loaded. (E) The indicated cell lines were immunostained for surface EphA2 or EphA5 expression and analyzed by flow cytometry. Gray histograms represent isotype controls. (F) The indicated cell lines were infected with KSHV in triplicate, and the infection rate was quantified by flow cytometry at 2 days postinfection. Data from a representative experiment are shown. (G) The 3×Flag expression histograms of infected 3×Flag-tagged ephrin receptor-transduced cell lines were divided into five successive gates, as shown. The infection rate within each gate was plotted against the fold MFI over the isotype of each gate. *, P < 0.05.

Article Snippet: Heparan sulfate antibody (F58-10E4) was purchased from Amsbio; integrin α3 antibody (P1B5) was purchased from Calbiochem; integrin αV, integrin β7, EphA2, and EphA5 antibodies (MAB12191, MAB4669, AF3035, and MAB541, respectively) were obtained from R&D Systems; integrin β1 and integrin β3 antibodies (T2S/16 and PM6/13, respectively) were obtained from Novus Biologicals; integrin β5 and EphA2 antibodies (AST-3T and SHM16, respectively) were obtained from BioLegend; xCT and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies (ab37185 and ab181602, respectively) were obtained from Abcam; DC-SIGN antibody (DCN47.5) was obtained from Miltenyi Biotec; EphA4 antibody (4C8H5) was obtained from ThermoFisher; EphB2 antibody (2D12C6) was obtained from Santa Cruz Biotech; and Flag antibody (M2) was obtained from Sigma-Aldrich.

Techniques: Infection, Construct, Sterility, Plasmid Preparation, Control, Expressing, Flow Cytometry, SDS Page

Journal: Journal of Virology

Article Title: A Kaposi's Sarcoma-Associated Herpesvirus Infection Mechanism Is Independent of Integrins α3β1, αVβ3, and αVβ5

doi: 10.1128/JVI.00803-18

Figure Lengend Snippet: The EphA2 ectodomain is sufficient to rescue the rate of infection of EPHA2 KO cells. (A) Diagram of EphA2 truncation and domain swap constructs. (B and C) WT, EPHA2 KO, and EPHA2 KO cells transduced with the EphA2 constructs indicated in panel A were infected with KSHV in triplicate, and the infection rate was quantified by flow cytometry at 2 days postinfection. The infection rates were normalized to the average rate of infection of WT cells, and data from a representative experiment are shown. (D) WT, EPHA2 KO, and the indicated transduced EPHA2 KO Caki-1 cells were immunostained for surface EphA2 expression and analyzed by flow cytometry. Gray histograms represent the isotype controls. *, P < 0.05.

Article Snippet: Heparan sulfate antibody (F58-10E4) was purchased from Amsbio; integrin α3 antibody (P1B5) was purchased from Calbiochem; integrin αV, integrin β7, EphA2, and EphA5 antibodies (MAB12191, MAB4669, AF3035, and MAB541, respectively) were obtained from R&D Systems; integrin β1 and integrin β3 antibodies (T2S/16 and PM6/13, respectively) were obtained from Novus Biologicals; integrin β5 and EphA2 antibodies (AST-3T and SHM16, respectively) were obtained from BioLegend; xCT and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies (ab37185 and ab181602, respectively) were obtained from Abcam; DC-SIGN antibody (DCN47.5) was obtained from Miltenyi Biotec; EphA4 antibody (4C8H5) was obtained from ThermoFisher; EphB2 antibody (2D12C6) was obtained from Santa Cruz Biotech; and Flag antibody (M2) was obtained from Sigma-Aldrich.

Techniques: Infection, Construct, Transduction, Flow Cytometry, Expressing

Journal: Nature Communications

Article Title: Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation

doi: 10.1038/s41467-020-15173-4

Figure Lengend Snippet: a FGF4 is a key signal that promotes differentiation of pluripotent mESCs. b Workflow for phosphoproteomic analysis of FGF4 signalling in Fgf4 −/− mESCs. Volcano plot showing significantly modified phosphosites after stimulation of Fgf4 −/− mESCs with FGF4 for 5 min ( c ) and 20 min ( d ). Phosphosites on known FGF4 pathway components are highlighted. e Protein kinase phosphopeptides that are significantly upregulated (>2-fold) on at least one time point (5 or 20 min) compared with control. Data are presented as mean ± SD ( n = 3). f Fgf4 −/− mESCs were stimulated with FGF4 for the indicated time, and EPHA2 pS898 and EPHA2 levels determined by immunoblotting. Source data are provided as a Source Data file.

Article Snippet: Cells were seeded on gelatin-coated coverslips, and fixed with PBS 4% PFA [w/v], permeabilised in PBS 0.5% Triton X-100 [v/v] for 5 min at room temperature, blocked with 3% bovine serum albumin [w/v] in PBS and incubated with EPHA2 antibody (R&D Systems) at 1:200 in blocking buffer for 2 h at room temperature.

Techniques: Modification, Control, Western Blot

Journal: Nature Communications

Article Title: Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation

doi: 10.1038/s41467-020-15173-4

Figure Lengend Snippet: a Average protein copy number per cell determined for receptor kinases in mESCs, using quantitative whole-cell proteomics. Data are presented as mean ± SD ( n = 3). b Workflow for quantification of EPH–EFN interactions in mESCs by EFN ligand affinity purification mass spectrometry. c Proof-of-principle identification of EPH–EFN interactions by EFN ligand affinity purification. EPHA2 levels were determined by immunoblotting. d Coomassie staining of EFNA1/B1 affinity purification from mESCs. EFNA1, EFNB1 and EPHA2 proteins are indicated. (*) = non-specific band. e Mass-spectrometry analysis of 75–130-kDa region of the Coomassie stained EFNA1/EFNB1 affinity purification shown in ( d ). Total spectral counts recovered for each EPH receptor family member are indicated. f EFNA1/B1 affinity purification from intact Epha2 +/+ and pooled Epha2 −/− mESCs. Phosphotyrosine (pTyr), EPHA2 and ERK1/2 levels were determined by immunoblotting. Note that the pTyr signal is specific for EPHA2, and is not detected in the absence of EFN ligand. g EPHA2 was immunoprecipitated from Epha2 +/+ and Epha2 −/− mESCs, and pTyr, EPHA2, EFNA1 and ERK1/2 levels determined by immunoblotting. h Epha2 +/+ mESCs were stimulated with EFNA1-expressing Epha2 −/− mESCs for 15 or 40 min. EPHA2 was immunoprecipitated, and pTyr and EPHA2 levels determined by immunoblotting. Source data are provided as a Source Data file.

Article Snippet: Cells were seeded on gelatin-coated coverslips, and fixed with PBS 4% PFA [w/v], permeabilised in PBS 0.5% Triton X-100 [v/v] for 5 min at room temperature, blocked with 3% bovine serum albumin [w/v] in PBS and incubated with EPHA2 antibody (R&D Systems) at 1:200 in blocking buffer for 2 h at room temperature.

Techniques: Affinity Purification, Mass Spectrometry, Western Blot, Staining, Immunoprecipitation, Expressing

Journal: Nature Communications

Article Title: Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation

doi: 10.1038/s41467-020-15173-4

Figure Lengend Snippet: a Epha2 +/+ , Epha2 −/− or Epha2 −/− mESCs (clone C4) stably expressing EPHA2 were cultured in the absence of LIF for 48 h. EPHA2, KLF4, DNMT3B, NANOG and OCT4 levels were determined by immunoblotting. b Epha2 +/+ or Epha2 −/− (clone C4) mESCs were maintained in 2i or differentiated in N2B27 media for 72 or 96 h, respectively, whereupon 10% of cells were replated in 2i. Total alkaline phosphatase staining is represented relative to Epha2 +/+ mESCs. The total number of alkaline phosphatase-positive colonies for Epha2 +/+ and Epha2 −/− mESCs is shown, and also represented relative to Epha2 +/+ mESCs. Data show mean ± SEM ( n = 3); statistical significance was determined using unpaired two-sided Student’s t test comparing Epha2 −/− with the Epha2 +/+ control (**** P < 0.0001, ** P = 0.0016). c Epha2 +/+ or Epha2 −/− (clone C4) mESCs stably expressing EFNA1, along with the respective parental controls, were grown in LIF/FBS, and KLF4, NANOG, EPHA2, EFNA1 and ERK1/2 levels determined by immunoblotting, or EPHA2 immunoprecipitated and pTyr and EPHA2 levels determined by immunoblotting. d Epha2 +/+ , Epha2 −/− or Epha2 −/− mESCs stably expressing EPHA2 were differentiated as embryoid bodies for 10 days, and the levels of Fgf5 , Brachyury , Mixl and Cer1 mRNA determined by qRT-PCR. Box-and-whisker plots show median, first and third quartiles, and maximum and minimum values. The results shown are for technical replicates from two independent experiments, including three Epha2 −/− clones ( n = 3); statistical significance at day 4 was determined using unpaired two-sided Student’s t test comparing each group with the Epha2 +/+ control (ns = not significant, * P = 0.0252, ** P = 0.0045, *** P < 0.0001). e Epha2 +/+ mESCs cultured in LIF/FBS were stimulated with 1 μg/ml clustered EFNA1 for the indicated times. ppERK1/2, total ERK1/2, STAT3 pY705 and total STAT3 levels were determined by immunoblotting. EPHA2 was immunoprecipitated, and pTyr and EPHA2 levels determined by immunoblotting. ppERK1/2 signal was quantified; data show mean ± SD ( n = 3); statistical significance was determined using one-sample two-sided t test comparing each group with control, theoretical mean = 1 (ns = not significant, 5 min; * P = 0.0467, 30 min; *** P = 0.0005, 45 min; ** P = 0.0014, 60 min; ** P = 0.0018). f Epha2 +/+ mESCs cultured in LIF/FBS were stimulated with 1 μg/ml clustered EFNA1 for the indicated times. SHP2 was immunoprecipitated, and SHP2 and EPHA2 levels detected by immunoblotting. Source data are provided as a Source Data file.

Article Snippet: Cells were seeded on gelatin-coated coverslips, and fixed with PBS 4% PFA [w/v], permeabilised in PBS 0.5% Triton X-100 [v/v] for 5 min at room temperature, blocked with 3% bovine serum albumin [w/v] in PBS and incubated with EPHA2 antibody (R&D Systems) at 1:200 in blocking buffer for 2 h at room temperature.

Techniques: Stable Transfection, Expressing, Cell Culture, Western Blot, Staining, Control, Immunoprecipitation, Quantitative RT-PCR, Whisker Assay, Clone Assay

Journal: Nature Communications

Article Title: Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation

doi: 10.1038/s41467-020-15173-4

Figure Lengend Snippet: a Diagram of potential phosphorylation sites within the EPHA2 S898 motif. Mass spectrometry analysis detects phosphorylation of at least three sites in EPHA2 immunoprecipitated from FGF4-stimulated Fgf4 −/− mESCs (see Supplementary Table ). b Fgf4 −/− mESCs were treated with 10 μM of the indicated inhibitors for 1 h, and stimulated with FGF4 for 10 min. EPHA2 pS898, EPHA2 and AKT pS473 levels were determined by immunoblotting. c Epha2 −/− mESCs were transfected with either wild type or 5E EPHA2 constructs, and stimulated with 1 μg/ml clustered EFNA1 for 15 min. EPHA2 was immunoprecipitated, and pTyr and EPHA2 levels determined by immunoblotting and quantified. Data show mean ± SD ( n = 4). d EPHA2 was immunoprecipitated from EPHA2 WT knock-in (KI) or 5A KI cell lines and pTyr and EPHA2 levels determined by immunoblotting (upper panel). Relative pTyr/EPHA2 signal was quantified (lower panel). Data show mean ± SD ( n = 4). e Phase-contrast images of EPHA2 WT KI or 5A KI mESC lines; scale bar = 100 µM. f EPHA2 WT KI or 5A KI cell lines were cultured in LIF/FBS medium for 48 h, and KLF4, NANOG, DNMT3B, OCT4 ppERK1/2 and ERK1/2 levels determined by immunoblotting. g Epha2 +/+ mESCs were differentiated as embryoid bodies for 10 days, and EPH receptor expression determined by qRT-PCR at the indicated time points. Box-and-whisker plots show median, first and third quartiles, and maximum and minimum values of four technical replicates ( n = 4). h Epha2 +/+ mESCs were differentiated as embryoid bodies for 10 days, and EFN ligand expression determined by qRT-PCR at the indicated time points. Box-and-whisker plots show median, first and third quartiles, and maximum and minimum values of four technical replicates ( n = 4). Epha2 ( i ) and Efna1 ( j ) mRNA expression in 2i mESCs undergoing differentiation in N2B27 was determined by qRT-PCR analysis at the indicated time points. Box-and-whisker plots show median, first and third quartiles, and maximum and minimum values of two technical and three biological replicates ( n = 3). Source data are provided as a Source Data file.

Article Snippet: Cells were seeded on gelatin-coated coverslips, and fixed with PBS 4% PFA [w/v], permeabilised in PBS 0.5% Triton X-100 [v/v] for 5 min at room temperature, blocked with 3% bovine serum albumin [w/v] in PBS and incubated with EPHA2 antibody (R&D Systems) at 1:200 in blocking buffer for 2 h at room temperature.

Techniques: Phospho-proteomics, Mass Spectrometry, Immunoprecipitation, Western Blot, Transfection, Construct, Knock-In, Cell Culture, Expressing, Quantitative RT-PCR, Whisker Assay

Journal: Nature Communications

Article Title: Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation

doi: 10.1038/s41467-020-15173-4

Figure Lengend Snippet: a Epha2 +/+ mESCs were transfected with the indicated siRNAs, and EPHA2, OCT4 and SOX2 levels determined by immunoblotting. A non-specific band was used as a loading control. b Epha2 and Oct4 mRNA expression was determined by qRT-PCR following transfection of Epha2 +/+ mESCs with control or OCT4 siRNA. Data show mean ± SEM of four technical replicates (n = 4). c Pluripotency transcription factor-binding sites in Epha2 gene regulatory regions were extracted from CODEX mESC ChIP-SEQ data ( http://codex.stemcells.cam.ac.uk ). d Epha2 +/+ mESCs were transfected with the indicated siRNAs, and stimulated with 1 μg/ml clustered EFNA1 for 15 min. EPHA2 was immunoprecipitated using EPHA2 antibody, and pTyr and EPHA2 levels determined by immunoblotting. e EFNA1/B1 affinity purification from control, dasatinib-treated or siOct4-transfected mESCs. pTyr, EPHA2, OCT4 and ERK1/2 levels were determined by immunoblotting. Source data are provided as a Source Data file.

Article Snippet: Cells were seeded on gelatin-coated coverslips, and fixed with PBS 4% PFA [w/v], permeabilised in PBS 0.5% Triton X-100 [v/v] for 5 min at room temperature, blocked with 3% bovine serum albumin [w/v] in PBS and incubated with EPHA2 antibody (R&D Systems) at 1:200 in blocking buffer for 2 h at room temperature.

Techniques: Transfection, Western Blot, Control, Expressing, Quantitative RT-PCR, Binding Assay, ChIP-sequencing, Immunoprecipitation, Affinity Purification

Journal: Nature Communications

Article Title: Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation

doi: 10.1038/s41467-020-15173-4

Figure Lengend Snippet: Expression (RPKM) of EPH ( a , b ) or EFN ( c , d ) mRNA in the inner cell mass (ICM) or trophectoderm (TE) of 64-cell mouse embryos ( a , c ) or 5-day human embryos ( b , d ). a , c n = 33 biologically independent cells for ICM, and n = 28 biologically independent cells for TE. b , d n = 73 biologically independent cells for ICM, and n = 142 biologically independent cells for TE. Box-and-whisker plots show median, first and third quartiles, and maximum and minimum values. e EPHA2 regulation and function in mESCs. In the pluripotent state, OCT4 and other pluripotency factors promote EPHA2 receptor expression, enabling activation by EFNA ligands to support pluripotency by restraining ERK1/2. During differentiation, FGF4 drives ERK1/2–RSK activity to phosphorylate and inhibit EPHA2, whilst ERK1/2 suppresses an OCT4–EPHA2 transcriptional module to disable EPHA2 receptor expression. Source data are provided as a Source Data file.

Article Snippet: Cells were seeded on gelatin-coated coverslips, and fixed with PBS 4% PFA [w/v], permeabilised in PBS 0.5% Triton X-100 [v/v] for 5 min at room temperature, blocked with 3% bovine serum albumin [w/v] in PBS and incubated with EPHA2 antibody (R&D Systems) at 1:200 in blocking buffer for 2 h at room temperature.

Techniques: Expressing, Whisker Assay, Activation Assay, Activity Assay