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: Journal of Molecular Cell Biology

Article Title: Inhibition of EphA2 by syndecan-4 in wounded skin regulates clustering of fibroblasts

doi: 10.1093/jmcb/mjae054

Figure Lengend Snippet: Expression of EphRs and cell repulsion are increased in the absence of SDC4. ( A ) Representative collisions between MEFs seeded on 5-μm fibronectin stripes (see and ). ( B ) Collisions were scored as ‘following’ if cells moved in the same direction after collision or ‘repulsion’ if cells moved in opposite directions ( n = 29). ( C ) Collisions, in the presence of 20 ng/ml TGFβ, were scored ( n = 34). ( D – F ) qPCR analysis of EphR and ephrin expression levels. Histograms depict fold changes relative to Sdc4 +/+ MEFs from a representative experiment, with n = 3 and experiments repeated up to 5 times. ( G – I ) Western blot analysis of EphA2 ( G , n = 3), EphA3 ( H , n = 3), and EphA4 ( I , n = 4) protein levels. ( J and K ) qPCR analysis of SDC4 and EphA2 mRNA levels ( J , n = 8) and western blot analysis of EphA2 protein level ( K , n = 3) in MEFs transfected with non-targeting (Control) or SDC4-targeting (SDC4) siRNA. Error bars represent standard error; significance tested by analysis of variance (ANOVA); * P < 0.05, ** P < 0.005, *** P < 0.0005.

Article Snippet: Protein was either immunoprecipitated with anti-EphA2 antibody (R&D Systems, AF639) and DynabeadsTM Protein G (ThermoFisher) before western blotting with DyLight800-conjugated streptavidin (ThermoFisher), for phosphotyrosine (4g10, Millipore) or pY596/pY602 EphA2 , or precipitated with DynabeadsTM Streptavidin (ThermoFisher) before western blotting for EphA2.

Techniques: Expressing, IF-cells, Western Blot, Transfection, Control

Journal: Journal of Molecular Cell Biology

Article Title: Inhibition of EphA2 by syndecan-4 in wounded skin regulates clustering of fibroblasts

doi: 10.1093/jmcb/mjae054

Figure Lengend Snippet: Reciprocal relationship between expression of SDC4 and surface expression of EphA2. ( A and B ) MEFs were surface-labelled with biotin before immunoprecipitating EphA2 and probing with fluorophore-conjugated streptavidin ( A , n = 3) or precipitating biotinylated proteins and blotting for EphA2 ( B , n = 7). ( C – E ) SDC4 was overexpressed in MEFs via retroviral infection with the SDC4 cDNA, using empty vector as a negative control. ( C ) SDC4 expression in infected MEFs by flow cytometry. ( D ) Western blot analysis of EphA2 protein levels in cell lysates ( n = 10). ( E ) Surface EphA2 protein levels by immunoprecipitating EphA2 from MEFs surface-labelled with biotin and probing with fluorophore-conjugated streptavidin ( n = 5). Error bars represent standard error; significance tested by ANOVA; * P < 0.05, ** P < 0.005, *** P < 0.0005.

Article Snippet: Protein was either immunoprecipitated with anti-EphA2 antibody (R&D Systems, AF639) and DynabeadsTM Protein G (ThermoFisher) before western blotting with DyLight800-conjugated streptavidin (ThermoFisher), for phosphotyrosine (4g10, Millipore) or pY596/pY602 EphA2 , or precipitated with DynabeadsTM Streptavidin (ThermoFisher) before western blotting for EphA2.

Techniques: Expressing, Retroviral, Infection, Plasmid Preparation, Negative Control, Flow Cytometry, Western Blot

Journal: Journal of Molecular Cell Biology

Article Title: Inhibition of EphA2 by syndecan-4 in wounded skin regulates clustering of fibroblasts

doi: 10.1093/jmcb/mjae054

Figure Lengend Snippet: Phosphorylation and endocytosis of EphA2 increase proportionally to expression. ( A and B ) MEFs were stimulated with 1 μg/ml clustered ephrinA1-Fc, followed by western blot analysis using antibody 4G10 for total phosphotyrosine ( A , n = 4) or an antibody against EphA2 residues Y596 and Y602 ( B , n = 4). Histograms represent phosphorylation levels normalised to EphA2. ( C and D ) EphA2 endocytosis in fibroblasts transfected with non-targeting (Control) or SDC4-targeting (SDC4) siRNA. ( C ) Flow cytometry analysis of SDC4 expression. ( D ) Fibroblasts were surface-labelled with biotin and stimulated with 1 μg/ml clustered ephrinA1-Fc. The internalised proteins were precipitated with streptavidin before blotting for EphA2 ( n = 6). ( E and F ) MEFs were stimulated with 1 μg/ml clustered ephrinA1-Fc before fixation and staining (see also ). ( E ) Representative images of MEFs stained for EEA1 (green), EphA2 (red), and counterstained with phalloidin (blue). Scale bar, 10 μm. ( F ) Pearson correlation analysis for colocalization between EphA2 and EEA1 ( n = 10). Error bars represent standard error; significance tested by ANOVA; * P < 0.05, ** P < 0.005, *** P < 0.0005.

Article Snippet: Protein was either immunoprecipitated with anti-EphA2 antibody (R&D Systems, AF639) and DynabeadsTM Protein G (ThermoFisher) before western blotting with DyLight800-conjugated streptavidin (ThermoFisher), for phosphotyrosine (4g10, Millipore) or pY596/pY602 EphA2 , or precipitated with DynabeadsTM Streptavidin (ThermoFisher) before western blotting for EphA2.

Techniques: Phospho-proteomics, Expressing, Western Blot, Transfection, Control, Flow Cytometry, Staining

Journal: Journal of Molecular Cell Biology

Article Title: Inhibition of EphA2 by syndecan-4 in wounded skin regulates clustering of fibroblasts

doi: 10.1093/jmcb/mjae054

Figure Lengend Snippet: Sdc4 –/– MEFs contract in response to ephrinA1. Sdc4 +/+ and Sdc4 –/– MEFs transfected with either non-targeting (Control) or EphA2-targeting (EphA2) siRNA were spread on 5 μg/ml fibronectin-coated dishes and stimulated with 1 μg/ml clustered ephrinA1-Fc. ( A ) Representative frames before and after the addition of ephrinA1-Fc (see – ). Scale bar, 20 μm. ( B ) Percentage of cells that contracted upon the addition of ephrinA1-Fc. The histogram represents the average of 3 experiments, with a total of 47 cells of each type scored. ( C ) Western blot analysis of EphA2 expression in cells used for the contraction assay and subsequent migration assay. Error bars indicate standard error; significance tested by ANOVA; *** P < 0.0005.

Article Snippet: Protein was either immunoprecipitated with anti-EphA2 antibody (R&D Systems, AF639) and DynabeadsTM Protein G (ThermoFisher) before western blotting with DyLight800-conjugated streptavidin (ThermoFisher), for phosphotyrosine (4g10, Millipore) or pY596/pY602 EphA2 , or precipitated with DynabeadsTM Streptavidin (ThermoFisher) before western blotting for EphA2.

Techniques: Transfection, Control, Western Blot, Expressing, Contraction Assay, Migration

Journal: Journal of Molecular Cell Biology

Article Title: Inhibition of EphA2 by syndecan-4 in wounded skin regulates clustering of fibroblasts

doi: 10.1093/jmcb/mjae054

Figure Lengend Snippet: SDC4 regulates EphA2 expression by activation of PKCα. ( A ) SDC4 cytodomain indicating the functions of residues to be tested. ( B – F ) Western blot analysis of EphA2 protein levels in Sdc4 +/+ MEFs, Sdc4 –/– MEFs, and Sdc4 –/– MEFs stably rescued with the indicated SDC4 cDNAs ( B , n = 5), Sdc4 +/+ ( C , n = 10) and Sdc4 –/– ( D , n = 4) MEFs treated daily with indicated concentrations of PMA for 5 days, Sdc4 +/+ MEFs treated daily with indicated concentrations of the PKCα inhibitor, BIM-1, for 5 days ( E , n = 6), and Sdc4 +/+ MEFs transfected with control or PKCα-targeting siRNA ( F , n = 6). Error bars represent standard error; significance tested by ANOVA; * P < 0.05, ** P < 0.005, *** P < 0.0005.

Article Snippet: Protein was either immunoprecipitated with anti-EphA2 antibody (R&D Systems, AF639) and DynabeadsTM Protein G (ThermoFisher) before western blotting with DyLight800-conjugated streptavidin (ThermoFisher), for phosphotyrosine (4g10, Millipore) or pY596/pY602 EphA2 , or precipitated with DynabeadsTM Streptavidin (ThermoFisher) before western blotting for EphA2.

Techniques: Expressing, Activation Assay, Western Blot, Stable Transfection, Transfection, Control

Journal: Journal of Molecular Cell Biology

Article Title: Inhibition of EphA2 by syndecan-4 in wounded skin regulates clustering of fibroblasts

doi: 10.1093/jmcb/mjae054

Figure Lengend Snippet: Homotypic repulsion of Sdc4 –/– MEFs is due to high EphA2 expression. ( A ) Representative collisions between Sdc4 +/+ ( n = 93) and Sdc4 –/– MEFs transfected with either non-targeting (Control, n = 78) or EphA2-targeting (EphA2, n = 14) siRNA (see – ). ( B ) Collisions were scored as ‘following’ or ‘repulsion’. ( C ) Time before both nuclei made a retrograde step post-collision. Boxes indicate median and 1st and 3rd quartiles and whiskers indicate data range. Significance tested by ANOVA; * P < 0.05.

Article Snippet: Protein was either immunoprecipitated with anti-EphA2 antibody (R&D Systems, AF639) and DynabeadsTM Protein G (ThermoFisher) before western blotting with DyLight800-conjugated streptavidin (ThermoFisher), for phosphotyrosine (4g10, Millipore) or pY596/pY602 EphA2 , or precipitated with DynabeadsTM Streptavidin (ThermoFisher) before western blotting for EphA2.

Techniques: Expressing, Transfection, Control

Journal: Journal of Molecular Cell Biology

Article Title: Inhibition of EphA2 by syndecan-4 in wounded skin regulates clustering of fibroblasts

doi: 10.1093/jmcb/mjae054

Figure Lengend Snippet: EphA2 expression in the fibroblasts of wounded skin is regulated by SDC4. ( A and B ) In mouse wound healing model, 4-mm full-thickness wounds were created on the backs of mice, and wound areas were recorded macroscopically. ( C – E ) qPCR analysis of EphA2 mRNA expression levels, relative to 18S internal control, in the bed of full-thickness skin wounds. Shown are relative expression changes over time in Sdc4 +/+ ( C ) and Sdc4 –/– ( D ) mice and direct comparison between two genotypes at 72 h ( E ) or 0 h ( F ) post-wounding ( n = 9). Error bars represent standard error; significance tested by ANOVA; * P < 0.05.

Article Snippet: Protein was either immunoprecipitated with anti-EphA2 antibody (R&D Systems, AF639) and DynabeadsTM Protein G (ThermoFisher) before western blotting with DyLight800-conjugated streptavidin (ThermoFisher), for phosphotyrosine (4g10, Millipore) or pY596/pY602 EphA2 , or precipitated with DynabeadsTM Streptavidin (ThermoFisher) before western blotting for EphA2.

Techniques: Expressing, Control, Comparison

Journal: Journal of Oncology

Article Title: Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer

doi: 10.1155/2009/951917

Figure Lengend Snippet: In vitro characterization of IgG25 and IgG28. (a) FACS-based whole cell binding assay was performed with IgG25 and IgG28 on human MiaPaCa2 and (b) on mouse MC38-CEA cells to determine the apparent Kd. Mean Fluorescence Intensities (MFIs) obtained with IgG25 and IgG28 over the logarithm of their molar concentration (LogM) are reported. (c) Binding competition experiments of IgG25 and IgG28 with ephrinA1 on MiaPaCa2 cells, a control isotypic IgG1 antibody (Ctrl IgG) was used as negative control. IgG25 (filled circles), IgG28 (empty circles), Ctrl IgG (filled triangles). (d) EphA2 immunoprecipitation from lysates of cells treated with Ctrl IgG, IgG25, IgG28 and ephA1-Fc, followed by Western Blot with antiphosphotyrosine antibody. After stripping, the same filter was probed with EphA2 antibody as loading control. (e) FACS-based whole cell binding with IgG25 and IgG28 on mouse N2A cells transiently transfected with expression vectors coding for members of Eph A receptor family (EphA1, EphA2, EphA3, EphA4, EphA5, and EphA7).

Article Snippet: To generate the stable Hek293 cell line expressing EphA2 in an inducible manner, 293/EphA2, the human EphA2 cDNA (NM_004431) was cloned from Origene Full Length Clone into the inducible expression vector pCEPTetO-MCS.

Techniques: In Vitro, Cell Binding Assay, Fluorescence, Concentration Assay, Binding Assay, Control, Negative Control, Immunoprecipitation, Western Blot, Stripping Membranes, Transfection, Expressing

Journal: Journal of Oncology

Article Title: Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer

doi: 10.1155/2009/951917

Figure Lengend Snippet: IgG25- and IgG28-mediated EphA2 internalization and degradation in MiaPaCa2 cells. (a) EphA2 internalization in response to IgG25, IgG28, and ephrinA1-Fc treatment. mAbs and ephrinA1-Fc labeled in red while cell nuclei in blue. Images were acquired at 20× magnification. Localization was revealed 1 hour after incubation on cells either at 4°C or at 37°C. (b) Time course Western blot analysis of EphA2 degradation after treatment with control IgG, IgG25, IgG28, and ephrinA1-Fc; anti- β -actin was used as loading control. (c) Densitometric analysis of the level of EphA2 expression measured by Western Blot in cells treated with control IgG (asterisks), IgG25 (squares), IgG28 (triangles), and ephrinA1-Fc (circles). Data are expressed as percentage of EphA2 expression over time.

Article Snippet: To generate the stable Hek293 cell line expressing EphA2 in an inducible manner, 293/EphA2, the human EphA2 cDNA (NM_004431) was cloned from Origene Full Length Clone into the inducible expression vector pCEPTetO-MCS.

Techniques: Labeling, Incubation, Western Blot, Control, Expressing

Journal: Journal of Oncology

Article Title: Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer

doi: 10.1155/2009/951917

Figure Lengend Snippet: EphA2 downstream signaling in MiaPaCa2 cells treated with IgG25 and IgG28. Cell lysates of MiaPaCa2 were assayed by Western Blotting with antiphospho-Akt, antiphospho-ERK, and anti-phospho FAK (Tyr 576). Antibodies directed to total Akt, total FAK, and actin were used as loading controls.

Article Snippet: To generate the stable Hek293 cell line expressing EphA2 in an inducible manner, 293/EphA2, the human EphA2 cDNA (NM_004431) was cloned from Origene Full Length Clone into the inducible expression vector pCEPTetO-MCS.

Techniques: Western Blot

Journal: Journal of Oncology

Article Title: Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer

doi: 10.1155/2009/951917

Figure Lengend Snippet: Effect of EphA2 antibodies on angiogenesis. (a) Representative CD31 immunostaining of paraffin embedded tumor section (magnification bar = 20 m). (b) Quantitative analysis of CD31 staining in tumors treated with control IgG, IgG25, and IgG28. The average data obtained from the analyses of two tumors selected from each group are reported as the percentage of CD31 + area in each entire section. In all panels, asterisks indicate statistically significant differences with respect to the control group (Student's t -test; P <.05).

Article Snippet: To generate the stable Hek293 cell line expressing EphA2 in an inducible manner, 293/EphA2, the human EphA2 cDNA (NM_004431) was cloned from Origene Full Length Clone into the inducible expression vector pCEPTetO-MCS.

Techniques: Immunostaining, Staining, Control

Journal: Journal of Oncology

Article Title: Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer

doi: 10.1155/2009/951917

Figure Lengend Snippet: EphA2 protein expression in tumors from treated mice. (a) Western Blot analysis with anti-EphA2 antibodies of lysates from five tumors treated with control IgG, with IgG25, or six tumors treated with IgG28. (b) Densitometric analysis of the ratio between EphA2 levels revealed by anti-EphA2 antibody in nonsaturating conditions and actin expression measured with antiactin antibody. Data on y axis are expressed as arbitrary units.

Article Snippet: To generate the stable Hek293 cell line expressing EphA2 in an inducible manner, 293/EphA2, the human EphA2 cDNA (NM_004431) was cloned from Origene Full Length Clone into the inducible expression vector pCEPTetO-MCS.

Techniques: Expressing, Western Blot, Control

Journal: Journal of Oncology

Article Title: Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo Efficacy in Pancreatic Cancer

doi: 10.1155/2009/951917

Figure Lengend Snippet: IgG25 and IgG28 modulate in vivo EphA2 downstream signaling: Western Blot analyses of tumor lysates with antiphospho FAK (Tyr 576) and anti-phospho Akt. Antibodies to total Akt, total FAK, and actin were used as loading controls. P values were calculated with respect to average densitometric value of group treated with control IgG.

Article Snippet: To generate the stable Hek293 cell line expressing EphA2 in an inducible manner, 293/EphA2, the human EphA2 cDNA (NM_004431) was cloned from Origene Full Length Clone into the inducible expression vector pCEPTetO-MCS.

Techniques: In Vivo, Western Blot, Control