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: Cancer Research

Article Title: Disruption of EphA2 Receptor Tyrosine Kinase Leads to Increased Susceptibility to Carcinogenesis in Mouse Skin

doi: 10.1158/0008-5472.can-06-0004

Figure Lengend Snippet: Figure 1. Characterization of EphA2 expression in mouse skin and role of EphA2 in tumor development. A, expression of EphA2 in skin extracts from wild-type and EphA2 knockout mice. Wild-type and EphA2 mutant mice at P8 were sacrificed. Skin tissues were removed from similar regions and snap frozen in liquid nitrogen. The frozen tissues were ground, lysed in radioimmunoprecipitation assay buffer, and subjected to immunoblot with rabbit polyclonal anti-EphA2 antibodies. The same membrane was reprobed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as loading control. B, expression of h-gal in EphA2 KO mouse skin. Frozen sections were fixed and stained with X-gal followed by counterstaining with nuclear Fast-Red. Bar, 40 Am. C, EphA2 knockout mice are more susceptible to DMBA/TPA two-stage chemical carcinogenesis compared to wild-type mice. Pictures were taken 13 weeks after DMBA initiation.

Article Snippet: The sections were then incubated with goat polyclonal antibodies that recognize the ectodomain of mouse EphA2 (R&D Systems, Minneapolis, MN) and rabbit polyclonal anti-ephrin-A1 (Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature for 1 hour followed by detection with donkey anti-goat IgG-FITC and donkey anti-rabbit IgG-Red X (Jackson ImmunoResearch Laboratories, West Grove, PA) at room temperature for 30 minutes.

Techniques: Expressing, Knock-Out, Mutagenesis, Radio Immunoprecipitation, Western Blot, Membrane, Control, Staining

Journal: Cancer Research

Article Title: Disruption of EphA2 Receptor Tyrosine Kinase Leads to Increased Susceptibility to Carcinogenesis in Mouse Skin

doi: 10.1158/0008-5472.can-06-0004

Figure Lengend Snippet: Figure 3. Malignant progression of DMBA/TPA–induced tumors in wild-type and EphA2 mutant mice. A, TPA treatment was stopped at 16 weeks after DMBA initiation. Tumors were collected 10 weeks later and processed for staining with H&E (a-e), anti-keratin 14 (f-g), or X-gal (i). a, H&E section of benign lesion from a wild-type (+/+) mouse. b, an invasive carcinoma from a wild-type mouse. c to d, H&E section of an invasive squamous cell carcinoma from a knockout mouse (/). e, H&E section of a spindle cell carcinoma from a knockout mouse. f, keratin 14 staining of an invasive EphA2/ carcinoma showing spindle cells infiltrating into tumor stroma. Bar, 40 Am. B, tumors arising in EphA2 knockout exhibited higher incidence of malignant conversion. Histopathologic examinations were done on the indicated numbers of tumors from each genotype. Tumors with clear evidence of invasive growth were quantitated. The proportion of invasive tumors was significantly different between EphA2+/+

Article Snippet: The sections were then incubated with goat polyclonal antibodies that recognize the ectodomain of mouse EphA2 (R&D Systems, Minneapolis, MN) and rabbit polyclonal anti-ephrin-A1 (Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature for 1 hour followed by detection with donkey anti-goat IgG-FITC and donkey anti-rabbit IgG-Red X (Jackson ImmunoResearch Laboratories, West Grove, PA) at room temperature for 30 minutes.

Techniques: Mutagenesis, Staining, Knock-Out

Journal: Cancer Research

Article Title: Disruption of EphA2 Receptor Tyrosine Kinase Leads to Increased Susceptibility to Carcinogenesis in Mouse Skin

doi: 10.1158/0008-5472.can-06-0004

Figure Lengend Snippet: Figure 5. Compartmentalized expression of EphA2 and ephrin-A1 in mouse skin. A, mice were sacrificed 16 weeks after DMBA initiation. Frozen sections were obtained from tumor-free areas of DMBA/TPA–treated mouse skin and were costained with a goat polyclonal antibody that recognizes EphA2 ectodomain and a rabbit polyclonal anti-ephrin-A1 antibody. Arrows, expression of ephrin-A1 and EphA2 in opposite gradient in outer root shaft; arrowheads, compartmentalized expression of ephrin-A1 and EphA2 in hair bulb. B, insets from (A) showing basal-suprabasal gradient expression of EphA2 and basal expression of ephrin-A1 in epidermis. C, EphA2 and ephrin-A1 expression in untreated normal skin from wild-type mice. Note the thinner epidermal layer in untreated skin compared with thickened epidermis in DMBA/TPA treatment skin shown in (B). D and E, EphA2 was up-regulated during skin tumorigenesis. Skin tumors were dissected and frozen sections with normal skin attached were cut. Tumors from EphA2+/ mice were stained with X-gal (D), whereas tumors from EphA2+/+ mice were stained with a goat polyclonal antibody that recognizes the ectodomain of EphA2 (E). Arrow, normal skin; arrowhead, papilloma. F, representative papillomas from wild-type mice stained for EphA2 and ephrin-A1. Arrows, tumor cells abutting the basement membrane. Bars, 40 Am.

Article Snippet: The sections were then incubated with goat polyclonal antibodies that recognize the ectodomain of mouse EphA2 (R&D Systems, Minneapolis, MN) and rabbit polyclonal anti-ephrin-A1 (Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature for 1 hour followed by detection with donkey anti-goat IgG-FITC and donkey anti-rabbit IgG-Red X (Jackson ImmunoResearch Laboratories, West Grove, PA) at room temperature for 30 minutes.

Techniques: Expressing, Staining, Membrane

Journal: Cancer Research

Article Title: Disruption of EphA2 Receptor Tyrosine Kinase Leads to Increased Susceptibility to Carcinogenesis in Mouse Skin

doi: 10.1158/0008-5472.can-06-0004

Figure Lengend Snippet: Figure 6. EphA2 kinase is required for ephrin-A1-induced inhibition of ERK1/2 MAPK and growth suppression in primary keratinocytes. A, EphA kinase activation by ephrin-A1 in EphA2++ and EphA2 +/ but not EphA2/ keratinocytes. Keratinocytes were isolated from newborn (P1) mice and cultured on Matrigel-coated culture plates. They were stimulated with 1.0 Ag/mL of ephrin-A1-Fc for 10 minutes. The unstimulated cells were used as control. EphA receptors, including EphA2, were precipitated from the cell lysates using ephrin-A1-Fc and were subjected to immunoblot analysis with rabbit polyclonal anti-phospho-EphA/B antibody. Membrane was stripped and blotted again for total EphA2. B, ephrin-A1 stimulation leads to inactivation of ERK1/2 MAPK in EphA2 +/+ and EphA2 +/ but not EphA2/

Article Snippet: The sections were then incubated with goat polyclonal antibodies that recognize the ectodomain of mouse EphA2 (R&D Systems, Minneapolis, MN) and rabbit polyclonal anti-ephrin-A1 (Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature for 1 hour followed by detection with donkey anti-goat IgG-FITC and donkey anti-rabbit IgG-Red X (Jackson ImmunoResearch Laboratories, West Grove, PA) at room temperature for 30 minutes.

Techniques: Inhibition, Activation Assay, Isolation, Cell Culture, Control, Western Blot, Membrane

Journal: Biology of Reproduction

Article Title: Expression and functional analyses of ephrin type-A receptor 2 in mouse spermatogonial stem cells ^†

doi: 10.1093/biolre/ioz156

Figure Lengend Snippet: Analysis of EPHA2 expression in spermatogonia. (A, B) RT-PCR analyses of Eph family genes in CDH1-selected testis cells (A) and GS cells (B). (C) Double immunostaining of 7-day-old pup and 6-week-old adult mouse testes using antibodies against EPHA2 and GFRA1, CDH1, or KIT. At least 67 cells (pup) or 52 cells (adult) in 10 fields were counted for each antigen. Arrows and arrowheads indicate cells expressing GFRA1 and CDH1, respectively. (D) Immunostaining of adult human testis using antibodies against EPHA2 and GFRA1. (E) Immunostaining of adult mouse testis using antibodies against EFNA1 and GATA4. Bar = 20 μm (C–E). Asterisk indicates statistical significance (P < 0.05).

Article Snippet: For MACS, dissociated testis cells (2 × 10 7 for wild-type and green mice; 5–10 × 10 7 for ROSA26 mice) were incubated with anti-EPHA2 antibody (5 μg/ml; MAB639; R&D systems, Minneapolis, MN) or anti-cadherin 1 antibody (5 μg/ml; ECCD2, gift from Dr. Masatoshi Takeichi from RIKEN CDB, Kobe, Japan) for 10 min at 4 °C.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Double Immunostaining, Immunostaining

Journal: Biology of Reproduction

Article Title: Expression and functional analyses of ephrin type-A receptor 2 in mouse spermatogonial stem cells ^†

doi: 10.1093/biolre/ioz156

Figure Lengend Snippet: Enrichment of mouse SSCs from the adult testis by MACS or FACS. (A) Cell recovery after MACS using anti-CDH1 or anti-EPHA2 antibodies (n = 3). (B) Macroscopic appearance of the recipient testis showing colonization of green mouse testis cells selected by MACS using an anti-EPHA2 antibody. (C) Colony count (n = 18 for EPHA2; n = 16 for control). (D) Fractionation of CDH1-selected ROSA mouse testis cells according to EPHA2 expression levels. Cells in the spermatogonia gate were divided into three populations (EPHA2negative, EPHA2low, and EPHA2high). (E) Real-time PCR analysis of spermatogonia genes (n = 3). (F) Macroscopic appearance of recipient testis transplanted with the EPHA2high population. The three populations of cells were transplanted into the seminiferous tubules of busulfan-treated mouse testes. (G) Colony count (n = 12). (H) Histological appearance of recipient testis showing normal spermatogenesis. Bar = 1 mm (B, F), 10 μm (H). Asterisk indicates statistical significance (P < 0.05).

Article Snippet: For MACS, dissociated testis cells (2 × 10 7 for wild-type and green mice; 5–10 × 10 7 for ROSA26 mice) were incubated with anti-EPHA2 antibody (5 μg/ml; MAB639; R&D systems, Minneapolis, MN) or anti-cadherin 1 antibody (5 μg/ml; ECCD2, gift from Dr. Masatoshi Takeichi from RIKEN CDB, Kobe, Japan) for 10 min at 4 °C.

Techniques: Cell Recovery, Control, Fractionation, Expressing, Real-time Polymerase Chain Reaction

Journal: Biology of Reproduction

Article Title: Expression and functional analyses of ephrin type-A receptor 2 in mouse spermatogonial stem cells ^†

doi: 10.1093/biolre/ioz156

Figure Lengend Snippet: Impaired proliferation and increased apoptosis by Epha2 KD in GS cells. (A) Flow cytometric analysis of EPHA2 expression after cytokine (FGF2 and GDNF) supplementation. Cells were cultured without cytokines for 2 days (NF) and then restimulated with FGF2 and GDNF (FG). Cells were analyzed on the next day after cytokine stimulation (n = 3). (B) Flow cytometric analysis of EPHA2 expression of GS cells 5 days after Epha2 KD (n = 3). (C) Cell recovery after Epha2 KD on MEFs. Cells were recovered 6 days after transfection (n = 12). (D) Cell recovery after Epha2 KD on laminin-coated plates (n = 9). GS cells were transfected with shRNA against Epha2, and cells were plated on laminin 2 days after transfection. Cells were recovered 2.5 h after plating. The relative cell recovery from the initially plated cells is indicated. (E) Flow cytometric analysis of ITGA6 expression after Epha2 KD. Fluorescence intensity was determined on the next day after transfection (n = 3). (F) Quantification of proliferating cells by MKI67 staining with at least 528 cells in 14 fields counted. Cells were recovered 4 days after transfection. (G) Quantification of apoptotic cells by TUNEL staining. TUNEL-stained cells were counterstained with Hoechst 33342 with at least 636 cells were counted. Cells were recovered 4 days after transfection. Scramble shRNA was used as a control in all KD experiments Bar = 50 μm (F, G). Asterisk indicates statistical significance (P < 0.05).

Article Snippet: For MACS, dissociated testis cells (2 × 10 7 for wild-type and green mice; 5–10 × 10 7 for ROSA26 mice) were incubated with anti-EPHA2 antibody (5 μg/ml; MAB639; R&D systems, Minneapolis, MN) or anti-cadherin 1 antibody (5 μg/ml; ECCD2, gift from Dr. Masatoshi Takeichi from RIKEN CDB, Kobe, Japan) for 10 min at 4 °C.

Techniques: Expressing, Cell Culture, Cell Recovery, Transfection, shRNA, Fluorescence, Staining, TUNEL Assay, Control

Journal: Biology of Reproduction

Article Title: Expression and functional analyses of ephrin type-A receptor 2 in mouse spermatogonial stem cells ^†

doi: 10.1093/biolre/ioz156

Figure Lengend Snippet: Analysis of EPHA2 and self-renewal factor receptors after Epha2 KD. (A) Western blot analysis of GS cells after Epha2 KD (n = 3). Cells were recovered 5–6 days after transfection. (B) Western blot analysis of GS cells after cytokine stimulation. Cells were cultured without cytokines for 2 days, and samples were collected on the next day after restimulation with FGF2 and GDNF. (C–E) Immunoprecipitation using antibodies against EPHA2 (C), RET (D), or FGFR2 (E).

Article Snippet: For MACS, dissociated testis cells (2 × 10 7 for wild-type and green mice; 5–10 × 10 7 for ROSA26 mice) were incubated with anti-EPHA2 antibody (5 μg/ml; MAB639; R&D systems, Minneapolis, MN) or anti-cadherin 1 antibody (5 μg/ml; ECCD2, gift from Dr. Masatoshi Takeichi from RIKEN CDB, Kobe, Japan) for 10 min at 4 °C.

Techniques: Western Blot, Transfection, Cell Culture, Immunoprecipitation

Journal: Biology of Reproduction

Article Title: Expression and functional analyses of ephrin type-A receptor 2 in mouse spermatogonial stem cells ^†

doi: 10.1093/biolre/ioz156

Figure Lengend Snippet: Functional analysis of Epha2 by spermatogonial transplantation. (A) Real-time PCR analysis of spermatogonial markers after Epha2 KD (n = 3). Cells were recovered 4 days after transfection, and the relative expression levels were determined by measuring the ratio between the experimental shRNA and control shRNA (indicated by the horizontal bar). (B) Cell recovery after transfection (n = 3). Cells were collected 2 days after transfection. (C) Real-time PCR analysis of Epha2 expression using GS cells (n = 3). GS cells were transfected by lentiviruses that express shRNA against Epha2. Cells were recovered on the next day after transfection. (D) Macroscopic appearance of recipient testis transplanted with pup testis cells after Epha2 KD or Epha2 CA OE. (E) Colony count (n = 17 for Epha2 KD; n = 18 for Epha2 CA). (F, G) Immuno- (F) or lectin (G) staining of recipient testes. Bar = 1 mm (D), 20 μm (F, G). Asterisk indicates statistical significance (P < 0.05).

Article Snippet: For MACS, dissociated testis cells (2 × 10 7 for wild-type and green mice; 5–10 × 10 7 for ROSA26 mice) were incubated with anti-EPHA2 antibody (5 μg/ml; MAB639; R&D systems, Minneapolis, MN) or anti-cadherin 1 antibody (5 μg/ml; ECCD2, gift from Dr. Masatoshi Takeichi from RIKEN CDB, Kobe, Japan) for 10 min at 4 °C.

Techniques: Functional Assay, Transplantation Assay, Real-time Polymerase Chain Reaction, Transfection, Expressing, shRNA, Control, Cell Recovery, Staining

Journal: Cell Adhesion & Migration

Article Title: EphA2 promotes cell adhesion and spreading of monocyte and monocyte/macrophage cell lines on integrin ligand-coated surfaces

doi: 10.1080/19336918.2015.1107693

Figure Lengend Snippet: Expression of endogenous and exogenous/dominant negative EphA2 in U937, EphA2ΔC-EGFP-U937, J774.1, and EphA2ΔC-EGFP-J774.1 cells. ( A ) Typical phase contrast and fluorescence micrographs highlighting the expression of the EphA2ΔC-EGFP protein. ( B ) EphA2 mRNAs amplified from the intracellular and extracellular regions by RT-PCR showing the expression of endogenous and total (endogenous plus exogenous) EphA2, respectively, in U937 and its subline. Densitometric quantification of the RT-PCR amplification levels of EphA2 from 3 independent experiments normalized to the levels of GAPDH. Data is presented as the mean ± SD. ** P < 0.01. ( C ) Amplification of endogenous and exogenous EphA2 in J774.1 cells and their subline cells by RT-PCR. Densitometric quantification of the RT-PCR amplification levels were determined from 3 independent experiments and normalized to the levels of GAPDH. Data is presented as the mean ± SD.

Article Snippet: The rat monoclonal PE-conjugated antibody against mouse EphA2 used in this study was from R&D Systems (233720).

Techniques: Expressing, Dominant Negative Mutation, Fluorescence, Amplification, Reverse Transcription Polymerase Chain Reaction

Journal: Cell Adhesion & Migration

Article Title: EphA2 promotes cell adhesion and spreading of monocyte and monocyte/macrophage cell lines on integrin ligand-coated surfaces

doi: 10.1080/19336918.2015.1107693

Figure Lengend Snippet: Representative histograms showing EphA2 and/or EGFP expression in U937 and EphA2ΔC-EGFP-U937 cells ( A ) as well as J774.1 and EphA2ΔC-EGFP-J774.1 cells ( B ).

Article Snippet: The rat monoclonal PE-conjugated antibody against mouse EphA2 used in this study was from R&D Systems (233720).

Techniques: Expressing

Journal: Cell Adhesion & Migration

Article Title: EphA2 promotes cell adhesion and spreading of monocyte and monocyte/macrophage cell lines on integrin ligand-coated surfaces

doi: 10.1080/19336918.2015.1107693

Figure Lengend Snippet: Molecular association of EphA2 with the β2 integrin/ICAM1 ( A ) and β2 integrin/VCAM1 complexes ( B ) in U937 and EphA2ΔC-EGFP-U937 cells.

Article Snippet: The rat monoclonal PE-conjugated antibody against mouse EphA2 used in this study was from R&D Systems (233720).

Techniques:

Journal: Cell Adhesion & Migration

Article Title: EphA2 promotes cell adhesion and spreading of monocyte and monocyte/macrophage cell lines on integrin ligand-coated surfaces

doi: 10.1080/19336918.2015.1107693

Figure Lengend Snippet: Primers and cycle numbers for PCR amplification used in U937 cells

Article Snippet: The rat monoclonal PE-conjugated antibody against mouse EphA2 used in this study was from R&D Systems (233720).

Techniques: Amplification

Journal: Cell Adhesion & Migration

Article Title: EphA2 promotes cell adhesion and spreading of monocyte and monocyte/macrophage cell lines on integrin ligand-coated surfaces

doi: 10.1080/19336918.2015.1107693

Figure Lengend Snippet: Primers and cycle numbers for PCR amplification used in J774.1 cells

Article Snippet: The rat monoclonal PE-conjugated antibody against mouse EphA2 used in this study was from R&D Systems (233720).

Techniques: Amplification

Journal: bioRxiv

Article Title: Contact inhibitory Eph signaling suppresses EGF-promoted cell migration by decoupling EGFR activity from vesicular recycling

doi: 10.1101/202705

Figure Lengend Snippet: (A,B) Representative immunofluorescence images (A) and quantification (B) of endogenous PM EGFR in Cos-7 cells ( N = 16-29 cells/condition) following stimulation with ephrinA1-Fc (A1, 2 μg/ml) for the indicated times (means ± sd). (C) Quantification of Akt activation by In-cell Western (ICW) in Cos-7 cells following A1 stimulation (left: 2 μg/ml; right: 15 min) (means ± s.e.m.). (D) Quantification of endogenous PM EGFR abundance by On-cell Western (OCW) and Akt activation in Cos-7 cells following treatment with the Akt inhibitor AktVIII (10 μM) for the times indicated (means ± s.e.m.). (E) EGF-Alexa647 binding (200 ng/ml, 2 min) to endogenous EGFR in Cos-7 cells as a measure of PM EGFR abundance following 1 h pretreatment with A1 (2 μg/ml), the PIKfyve inhibitor YM201636 (YM, 200 nM) or both ( N =33-59 cells/condition) (means ± sd). (F) Representative confocal images of Cos-7 cells expressing EGFR-mCherry before (top left) and after (top right) treatment with AktVIII (10 μM, 1 h) and quantification of the increase in endosomal EGFR-mCherry during AktVIII treatment (bottom, N = 6 cells, means ± sd). (G) Representative time-lapse confocal images of Cos-7 cells expressing EGFR-mCherry and EphA2-mCitrine following A1 stimulation (2 μg/ml). (H) Quantification of endosomal EGFR-mCherry and EphA2-mCitrine from time-lapse confocal imaging (G) during A1 stimulation ( N = 7 cells, means ± sd). (I) Quantification of PM EGFR-mCherry and EphA2-mCitrine abundance by OCW during A1 stimulation (2 μg/ml) (means ± s.e.m.). (J) Immunofluorescence measurements of EGFR intensity in Rab5-, Rab5/Rab7-and Rab7-positive endosomal compartments in control, A1-(2 μg/ml, 1 h) and AktVIII-(10 μM, 1 h) pretreated Cos-7 cells prior to (left) and following EGF stimulation (right, 100 ng/ml, 1 h). ( N =6-11 cells/condition). Data are represented by Tukey boxplots with the mean denoted as a cross and the median as a line. (K) Measurements of EGFR recycling in control and A1-pretreated cells (2 μg/ml, 1 h) by immunofluorescence prior to (pre), after EGF stimulation (10 ng/ml, 15 min) and 15 min following EGF washout ( N = 34-40 cells/condition, means ± sd). Statistical significance was determined in B, E, J and K using a one-way ANOVA with Sidak’s post-hoc test (***, p < 0.001). Scale bars = 20 μm.

Article Snippet: Mouse anti-Akt (2920, Cell Signaling Technology (CST), Danvers, MA, USA), mouse anti-Akt-Alexa488 (2917, CST), rabbit anti-phospho-Akt(Ser 473 ) (4060, CST), rabbit anti-phospho-Akt (Ser 473 )-Alexa647 (4075, CST), mouse anti-HA (9658, Sigma-Aldrich, St.Louis, MO, USA), rabbit anti-EGFR (4267, CST), goat anti-EGFR (AF231, R&D Systems, Minneapolis, MN, USA), mouse anti-phospho-EGFR(Tyr 845 ) (558381, BD Biosciences, Heidelberg, Germany), rabbit anti-phospho-EGFR(Tyr 1045 ) (2237, CST), mouse anti-phospho-EGFR(Tyr 1068 ) (2236, CST), goat anti-EphA2 (R&D Systems), rabbit anti-phospho-Eph(Tyr 588/596 ) (Abcam, Cambridge, UK), mouse anti-ERK1/2 (4696, CST), rabbit anti-phospho-ERK(Thr 202/Tyr204 ) (4370, CST), mouse anti-Rab5 (610724, BD Biosciences), rabbit anti-Rab7 (9367, CST), rabbit anti-phospho-Rb(Ser807/811, CST) mouse anti-tubulin (6074, Sigma-Aldrich)

Techniques: Immunofluorescence, Activation Assay, In-Cell ELISA, Western Blot, Binding Assay, Expressing, Imaging

Journal: bioRxiv

Article Title: Contact inhibitory Eph signaling suppresses EGF-promoted cell migration by decoupling EGFR activity from vesicular recycling

doi: 10.1101/202705

Figure Lengend Snippet: (A-D) Quantification of endogenous PM EGFR abundance by ICW following ephrinA1-Fc stimulation (A1, 2 μg/ml) of (A) HEK293, (B) NIH 3T3, (C) MCF10Aand (D) MDA-MB-231 cells. (means ± s.e.m) (E) Single cell immunofluorescence measurements of EGFR expression in the cell lines used in this study. (F-G) Cos-7 lysates immunoprecipitated (IP) with anti-EGFR (left) or blotted for total proteins (right) following stimulation with EGF (100 ng/ml) or A1 (2 μg/ml) for the indicated times. IP was probed with anti-HA (to detect co-transfected HA-ubiquitin), anti-pTyr 845 , anti-pTyr 1068 , anti-pTyr 1045 (to detect phosphorylated EGFR) and anti-EGFR. Total lysates were probed with anti phospho-Eph (pEph), anti-EphA2, anti-pTyr 1068 , anti-EGFR and anti-tubulin. Shown are (F) representative blots and (G) quantification of EGFR phosphorylation (pTyr 845 ) from four independent experiments (means ± s.e.m). Statistical significance was determined using a one-way ANOVA with Sidak’s post-hoc test (**, p < 0.01).

Article Snippet: Mouse anti-Akt (2920, Cell Signaling Technology (CST), Danvers, MA, USA), mouse anti-Akt-Alexa488 (2917, CST), rabbit anti-phospho-Akt(Ser 473 ) (4060, CST), rabbit anti-phospho-Akt (Ser 473 )-Alexa647 (4075, CST), mouse anti-HA (9658, Sigma-Aldrich, St.Louis, MO, USA), rabbit anti-EGFR (4267, CST), goat anti-EGFR (AF231, R&D Systems, Minneapolis, MN, USA), mouse anti-phospho-EGFR(Tyr 845 ) (558381, BD Biosciences, Heidelberg, Germany), rabbit anti-phospho-EGFR(Tyr 1045 ) (2237, CST), mouse anti-phospho-EGFR(Tyr 1068 ) (2236, CST), goat anti-EphA2 (R&D Systems), rabbit anti-phospho-Eph(Tyr 588/596 ) (Abcam, Cambridge, UK), mouse anti-ERK1/2 (4696, CST), rabbit anti-phospho-ERK(Thr 202/Tyr204 ) (4370, CST), mouse anti-Rab5 (610724, BD Biosciences), rabbit anti-Rab7 (9367, CST), rabbit anti-phospho-Rb(Ser807/811, CST) mouse anti-tubulin (6074, Sigma-Aldrich)

Techniques: Immunofluorescence, Expressing, Immunoprecipitation, Transfection

Journal: bioRxiv

Article Title: Contact inhibitory Eph signaling suppresses EGF-promoted cell migration by decoupling EGFR activity from vesicular recycling

doi: 10.1101/202705

Figure Lengend Snippet: (A) Quantification of Akt (top) and ERK (bottom) activation by ICW in control and ephrinA1-Fc-pretreated (A1, 2 μg/ml, 1 h) Cos-7 cells endogenously expressing the receptors following EGF stimulation (1 ng/ml) (means ± s.e.m.) (B) Quantification of Akt activation in control or A1-pretreated (2 μg/ml, 1 h) HEK293 cells, followed by 30 min treatment with the dynamin inhibitor dynole 34-2 (100 μM, top) or its negative control analog dynole 31-2 (100 μM, bottom), then stimulated with EGF (1 ng/ml) for the times indicated (means ± s.e.m.) (C) Quantification of EGF-promoted Akt activation in Cos-7 cells following pretreatment with increasing concentrations of A1 (0.02, 0.2 and 2 μg/ml, 1 h) (means ± s.e.m.) (D) Representative images of EphA2 activity using a FRET-based sensor (LIFEA2), whereby a decrease in fluorescence lifetime (τ, ns) represents an increase in activity, and fluorescence intensity measurements of LIFEA2-mCitrine and SH2-mCherry in Cos-7 cells. Scale bar = 20 μm. (E,F) Single cell measurements of Akt (E) and ERK (F) activation versus cell-cell contact in 2-D cultures (% cell circumference) in unstimulated and EGF stimulated (20 ng/ml, 1 h) Cos-7 cells. A sum-of-squares F test was used to determine significance: Akt, unstimulated: F = 16.0, p = 0.001, r 2 = 0.432; Akt, EGF: F= 21.4, p < 0.001, r 2 = 0.322; ERK, unstimulated: F = 0.180, p = 0.673, r 2 = 0.003; ERK, EGF: F = 0.321, p = 0.575, r 2 = 0.009.

Article Snippet: Mouse anti-Akt (2920, Cell Signaling Technology (CST), Danvers, MA, USA), mouse anti-Akt-Alexa488 (2917, CST), rabbit anti-phospho-Akt(Ser 473 ) (4060, CST), rabbit anti-phospho-Akt (Ser 473 )-Alexa647 (4075, CST), mouse anti-HA (9658, Sigma-Aldrich, St.Louis, MO, USA), rabbit anti-EGFR (4267, CST), goat anti-EGFR (AF231, R&D Systems, Minneapolis, MN, USA), mouse anti-phospho-EGFR(Tyr 845 ) (558381, BD Biosciences, Heidelberg, Germany), rabbit anti-phospho-EGFR(Tyr 1045 ) (2237, CST), mouse anti-phospho-EGFR(Tyr 1068 ) (2236, CST), goat anti-EphA2 (R&D Systems), rabbit anti-phospho-Eph(Tyr 588/596 ) (Abcam, Cambridge, UK), mouse anti-ERK1/2 (4696, CST), rabbit anti-phospho-ERK(Thr 202/Tyr204 ) (4370, CST), mouse anti-Rab5 (610724, BD Biosciences), rabbit anti-Rab7 (9367, CST), rabbit anti-phospho-Rb(Ser807/811, CST) mouse anti-tubulin (6074, Sigma-Aldrich)

Techniques: Activation Assay, Expressing, Negative Control, Activity Assay, Fluorescence

Journal: bioRxiv

Article Title: Contact inhibitory Eph signaling suppresses EGF-promoted cell migration by decoupling EGFR activity from vesicular recycling

doi: 10.1101/202705

Figure Lengend Snippet: Akt and ERK activation were quantified by ICW in (A) Cos-7 cells ectopically expressing EGFR and EphA2, and in (B) HEK293 cells endogenously expressing the receptors. Cells were pretreated with vehicle (control) or ephrinA1-Fc (A1, 2 μg/ml) for 1 h, followed by 1 ng/ml EGF stimulation for the indicated times. Data represent means ± s.e.m.

Article Snippet: Mouse anti-Akt (2920, Cell Signaling Technology (CST), Danvers, MA, USA), mouse anti-Akt-Alexa488 (2917, CST), rabbit anti-phospho-Akt(Ser 473 ) (4060, CST), rabbit anti-phospho-Akt (Ser 473 )-Alexa647 (4075, CST), mouse anti-HA (9658, Sigma-Aldrich, St.Louis, MO, USA), rabbit anti-EGFR (4267, CST), goat anti-EGFR (AF231, R&D Systems, Minneapolis, MN, USA), mouse anti-phospho-EGFR(Tyr 845 ) (558381, BD Biosciences, Heidelberg, Germany), rabbit anti-phospho-EGFR(Tyr 1045 ) (2237, CST), mouse anti-phospho-EGFR(Tyr 1068 ) (2236, CST), goat anti-EphA2 (R&D Systems), rabbit anti-phospho-Eph(Tyr 588/596 ) (Abcam, Cambridge, UK), mouse anti-ERK1/2 (4696, CST), rabbit anti-phospho-ERK(Thr 202/Tyr204 ) (4370, CST), mouse anti-Rab5 (610724, BD Biosciences), rabbit anti-Rab7 (9367, CST), rabbit anti-phospho-Rb(Ser807/811, CST) mouse anti-tubulin (6074, Sigma-Aldrich)

Techniques: Activation Assay, Expressing