Journal: JCI Insight

Article Title: MyD88/CD40 signaling retains CAR T cells in a less differentiated state

doi: 10.1172/jci.insight.136093

Figure Lengend Snippet: ( A ) Scheme of EphA2-CAR constructs. ( B ) Summary plot of %tCD19 + T cells ( n = 5, mean ± SEM, 1-way ANOVA with Tukey’s test for multiple comparisons). ( C ) Summary plot of %F(ab′) 2 -positive T cells ( n = 5, mean ± SEM, 1-way ANOVA with Tukey’s test for multiple comparisons). ( D ) CAR T cell production of Th1 (IFN-γ and IL-2) and Th2 (IL-4 and IL-10) cytokines after 24-hour coculture at a 2:1 ratio against EphA2-positive (LM7, U373 WT) and EphA2-negative (BV173, U373 EphA2 KO) cell lines or in media alone ( n = 5, mean ± SEM, 2-way ANOVA with Dunnett’s test for multiple comparisons, all statistical analysis is in comparison with NT cells). Dot colors: black, media; light gray, BV173 (EphA2 negative); dark gray, U373 EphA2 KO (EphA2 negative); dark blue: U373 (EphA2 positive); light blue, LM7 (EphA2 positive). ( E ) Summary plots of Th1 and Th2 cytokine production against EphA2-positive cell lines U373 and LM7 ( n = 5, mean ± SEM, values were log transformed before 2-way ANOVA with Tukey’s test for multiple comparisons). ( F ) CAR T cells were incubated with increasing amounts of tumor cells for 24 hours, and the remaining live tumor cells were quantified with an MTS assay (2-way ANOVA with Tukey’s test for multiple comparisons, mean ± SEM, LM7: n = 4, U373 WT and U373 EphA2 KO: n = 9). For LM7 and U373, asterisks refer to statistical comparison of MC-CAR with NT and CD28-CAR with MC-CAR. For U373 EphA2 KO, asterisks refer to statistical comparison of 41BB-CAR with NT and CD28-CAR with NT. # P < 0.1; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: After SDS-PAGE and wet transfer, the membrane was blocked with 5% milk in TBS-Tween (TBST), then incubated with primary antibodies: goat anti-human EphA2 (R&D Systems, Bio-Techne, AF3035), rabbit anti-human Bcl-2 (clone D55G8, Cell Signaling Technology), rabbit anti-human Myb (clone D2R4Y, Cell Signaling Technology), or mouse anti-human GAPDH (clone 0411, Santa Cruz Biotechnology).

Techniques: Construct, Comparison, Transformation Assay, Incubation, MTS Assay

Journal: JCI Insight

Article Title: MyD88/CD40 signaling retains CAR T cells in a less differentiated state

doi: 10.1172/jci.insight.136093

Figure Lengend Snippet: T cells were cocultured with tumor cells at a 2:1 ratio with weekly restimulation against fresh tumor cells until they lost their effector function and no longer killed all the tumor cells. ( A ) Average expansion of CAR T cells against EphA2-positive (U373 and LM7) and U373 EphA2 KO cell line (mean ± SEM, LM7: n = 4; U373: n = 8 [NT, CD28, MC], n = 4 [41BB]; U373 KO: n = 8 [NT, CD28, MC], n = 6 [41BB]). ( B ) Summary of the maximum expansion CAR T cells from individual donors achieved against EphA2-positive tumor cells and the maximum number of times CAR T cells were able to kill fresh EphA2-positive tumor cells ( n = 12 [NT, CD28, and MC], n = 8 [41BB]; median and quartiles, 1-way ANOVA with Tukey’s test for multiple comparisons). T cells were phenotyped 7 days after stimulation with U373. ( C ) Summary plot of CD4/CD8 composition after stimulation with U373 ( n = 3, mean ± SEM). ( D ) Scheme for phenotyping T cells and representative flow cytometry plots of CCR7 and CD45RA expression on CAR T cells after stimulation with U373. ( E ) Summary plot of T cell phenotype after stimulation with U373 ( n = 4, mean ± SEM, 2-way ANOVA with Tukey’s test for multiple comparisons). All statistical tests were performed in comparison with MC-CAR T cells (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001).

Article Snippet: After SDS-PAGE and wet transfer, the membrane was blocked with 5% milk in TBS-Tween (TBST), then incubated with primary antibodies: goat anti-human EphA2 (R&D Systems, Bio-Techne, AF3035), rabbit anti-human Bcl-2 (clone D55G8, Cell Signaling Technology), rabbit anti-human Myb (clone D2R4Y, Cell Signaling Technology), or mouse anti-human GAPDH (clone 0411, Santa Cruz Biotechnology).

Techniques: Flow Cytometry, Expressing, Comparison

Journal: JCI Insight

Article Title: MyD88/CD40 signaling retains CAR T cells in a less differentiated state

doi: 10.1172/jci.insight.136093

Figure Lengend Snippet: ( A – D ) NSG mice were injected with 1 × 10 6 LM7-ffLuc i.p. One week later, mice were injected with 1 × 10 4 or 1 × 10 5 CD28-, 41BB-, or MC-CAR T cells. PBS and 1 × 10 5 Delta-CAR T cells were used as controls. ( A ) Total flux from tumor cells in all mice treated with 1 × 10 4 EphA2 CAR T cells (PBS: n = 5; CD28, 41BB, MC: n = 10). ( B ) Total flux from tumor cells in all mice treated with 1 × 10 5 EphA2 CAR T cells (Delta: n = 5, CD28: n = 8, 41BB: n = 9, MC: n = 10). ( C and D ) Kaplan-Meier survival analysis of mice treated with 1 × 10 4 ( C ) or 1 × 10 5 ( D ) EphA2 CAR T cells (log-rank Mantel-Cox test with Bonferroni’s correction for multiple comparisons; * P < 0.05; ** P < 0.01; *** P < 0.001). Experiments were repeated twice with CAR T cells generated from 2 different healthy donors.

Article Snippet: After SDS-PAGE and wet transfer, the membrane was blocked with 5% milk in TBS-Tween (TBST), then incubated with primary antibodies: goat anti-human EphA2 (R&D Systems, Bio-Techne, AF3035), rabbit anti-human Bcl-2 (clone D55G8, Cell Signaling Technology), rabbit anti-human Myb (clone D2R4Y, Cell Signaling Technology), or mouse anti-human GAPDH (clone 0411, Santa Cruz Biotechnology).

Techniques: Injection, Generated

Journal: Nature Communications

Article Title: Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2

doi: 10.1038/ncomms8679

Figure Lengend Snippet: ( a ) Whole-cell lysates from HeLa cells treated with TNF-α (20 ng ml −1 ) for 10, 20 and 60 min were separated by Zn 2+ -Phos-tag SDS–PAGE and immunoblotted with anti-EphA2 and EGFR antibodies. ( b ) Whole-cell lysates from HeLa cells treated with TNF-α for 20 min were separated by Zn 2+ -Phos-tag SDS–PAGE and immunoblotted with anti-EphA2, pS-EphA2 and pY-EphA2. ( c ) Whole-cell lysates from HeLa cells treated with ephrin-A1 (100 ng ml −1 ) for 10 min or TNF-α for 20 min were separated by normal SDS–PAGE and immunoblotted with anti-pS-EphA2, pY-EphA2, EphA2 and α-tubulin antibodies. ( d ) HeLa cells were stimulated with TNF-α for the indicated periods. Whole-cell lysates were electrophoresed and probed with primary antibodies against pS-EphA2, pY-EphA2, EphA2, pT-EGFR, pS-EGFR, EGFR and α-tubulin. ( e ) HeLa cells were stimulated with TNF-α for 20 and 60 min. After fixation and permeabilization, cells were immunofluorescently stained with pS-EphA2, EphA2 or EGFR (clone LA1). Scale bar, 20 μm. Shown are representative images from three independent experiments.

Article Snippet: Antibodies against total EphA2 (C-20; sc-924), RSK1 (C-21; sc-231), RSK2 (C-19; sc-1430), EGFR (1005; sc-03), TAK1 (M-579; sc-7162), β-actin (I-19; sc-1616) and α-tubulin (B-7; sc-5286) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: SDS Page, Staining

Journal: Nature Communications

Article Title: Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2

doi: 10.1038/ncomms8679

Figure Lengend Snippet: ( a , b ) HeLa ( a ) or T98G ( b left) cells were pre-treated with LY294002 (10 μM) or MK-2206 (10 μM) for 30 min and then stimulated with TNF-α for 20 min. T98G cells were starved using FCS-free medium for 24 h, treated with LY294002 for 30 min and then treated with 10% FCS for 10 min ( b , right). ( c ) MDA-MB-231 and Panc-1 cells were treated with LY294002 for 30 min. ( d ) HeLa cells stably transfected shRNA expression vectors against luciferase and TAK1 were stimulated with TNF-α for 20 min. ( e ) HeLa cells were transfected with siRNAs against TAK1 or negative control. At 72 h post transfection, cells were treated with TNF-α for 20 min. Whole-cell lysates were immunoblotted with anti-pS-EphA2, EphA2, pAKT, pRSK, RSK1, RSK2, TAK1, β-actin and α-tubulin antibodies.

Article Snippet: Antibodies against total EphA2 (C-20; sc-924), RSK1 (C-21; sc-231), RSK2 (C-19; sc-1430), EGFR (1005; sc-03), TAK1 (M-579; sc-7162), β-actin (I-19; sc-1616) and α-tubulin (B-7; sc-5286) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Stable Transfection, Transfection, shRNA, Expressing, Luciferase, Negative Control

Journal: Nature Communications

Article Title: Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2

doi: 10.1038/ncomms8679

Figure Lengend Snippet: ( a ) HeLa cells were stimulated with TNF-α for the indicated periods. Whole-cell lysates were immunoblotted with anti-pS-EphA2, EphA2, pRSK, RSK1, RSK2 and α-tubulin antibodies. ( b , c ) Whole-cell lysates from HeLa cells pre-treated with LY294002 (10 μM), SB203580 (10 μM), U0126 (5 μM) or BI-D1870 (10 μM) for 30 min and then stimulated with TNF-α for 20 min were separated by Zn 2+ -Phos-tag SDS–PAGE and immunoblotted with anti-EphA2 and pS-EphA2 antibodies ( b ), or by normal SDS–PAGE and immunoblotted with anti-pS-EphA2, EphA2, pT-EGFR, pS-EGFR, EGFR, pRSK, RSK1, RSK2 and α-tubulin antibodies ( c ). ( d ) HeLa cells were pre-treated with LY294002 or BI-D1870 for 30 min and then stimulated with NaCl (0.3 M), TPA (100 ng ml −1 ) or EGF (10 ng ml −1 ) for 10 min. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, RSK1, RSK2, pAKT and α-tubulin. ( e ) T98G and U-87 MG cells starved in FCS-free medium for 24 h were treated with LY294002, MK-2206, U0126 and BI-D1870 for 30 min and then stimulated with 10% FCS for 10 min. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, RSK1, RSK2, pAKT, pERK and α-tubulin.

Article Snippet: Antibodies against total EphA2 (C-20; sc-924), RSK1 (C-21; sc-231), RSK2 (C-19; sc-1430), EGFR (1005; sc-03), TAK1 (M-579; sc-7162), β-actin (I-19; sc-1616) and α-tubulin (B-7; sc-5286) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: SDS Page

Journal: Nature Communications

Article Title: Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2

doi: 10.1038/ncomms8679

Figure Lengend Snippet: ( a , b ) HEK293 cells were transfected with expression vectors for EphA2, RSK1 and its substitution mutants. At 24 h post transfection, whole-cell lysates were immunoblotted with anti-pS-EphA2, pY-EphA2, EphA2, pRSK, RSK1 and α-tubulin antibodies. ( c ) HeLa cells were transfected with siRNAs against RSK1, RSK2 or negative control. At 72 h post transfection, cells were stimulated with TNF-α for 20 min. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, RSK1, RSK2 and α-tubulin. ( d ) Recombinant human GST-EphA2 was incubated with recombinant human active GST-RSK1 or RSK2 in the absence or presence of BI-D1870 (0.1 μM) at 30 °C for 30 min. The reaction mixtures were analysed by immunoblotting with anti-pS-EphA2, EphA2, RSK1 and RSK2 antibodies.

Article Snippet: Antibodies against total EphA2 (C-20; sc-924), RSK1 (C-21; sc-231), RSK2 (C-19; sc-1430), EGFR (1005; sc-03), TAK1 (M-579; sc-7162), β-actin (I-19; sc-1616) and α-tubulin (B-7; sc-5286) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Expressing, Negative Control, Recombinant, Incubation, Western Blot

Journal: Nature Communications

Article Title: Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2

doi: 10.1038/ncomms8679

Figure Lengend Snippet: ( a ) Whole-cell lysates from HeLa cells treated with TNF-α for 20 min or untreated MDA-MB-231 cells were separated by Zn 2+ -Phos-tag SDS–PAGE and immunoblotted with anti-EphA2 antibody. ( b – f ) MDA-MB-231 cells were pre-treated with BI-D1870 (10 μM) for 30 min and then scratched with a pipette tip. After 48 h of incubation, whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2 and β-actin ( b ) Migrated cells were counted manually under a microscope ( c ) Data are the means±s.d. of at least three fields. Similar results were obtained in at least three independent experiments. * P <0.05 by Student's t -test. At the same time, the migration border cells were immunofluorescently stained with anti-pS-EphA2 or EphA2 antibodies ( d , e ) and cells harbouring lamellipodia were counted manually under a microscope ( f ) Scale bar, 20 μm. Data are the means±s.d. of at least three fields. Similar results were obtained in at least three independent experiments. * P <0.05 by Student's t -test. ( g , h ) MDA-MB-231 cells were transfected with siRNA against EphA2 or negative control and EphA2 mutation-expression plasmids. The immunoblotting results from whole-cell lysates with anti-pS-EphA2, EphA2 and β-actin antibodies are shown in g and the results of scratch assay are shown in h . Data are the means±s.d. of at least three fields. Similar results were obtained in at least three independent experiments. * P <0.05 by analysis of variance followed by Tukey–Kramer HSD test.

Article Snippet: Antibodies against total EphA2 (C-20; sc-924), RSK1 (C-21; sc-231), RSK2 (C-19; sc-1430), EGFR (1005; sc-03), TAK1 (M-579; sc-7162), β-actin (I-19; sc-1616) and α-tubulin (B-7; sc-5286) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: SDS Page, Transferring, Incubation, Microscopy, Migration, Staining, Transfection, Negative Control, Mutagenesis, Expressing, Western Blot, Wound Healing Assay

Journal: Nature Communications

Article Title: Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2

doi: 10.1038/ncomms8679

Figure Lengend Snippet: ( a ) Human melanoma cells (A2058, SK-MEL-28, A375, UACC62, UACC257 and SK-MEL-2), ( b ) DLD-1 colon cancer cells and ( c ) lung adenocarcinoma cells (PC-9, HCC827, HCC4006, NCI-H1650, H2228 and A549) were treated with vemurafenib (1 μM), BI-D1870 (10 μM), gefitinib (1 μM), crizotinib (10 μM) or U0126 (5 μM) for 30–60 min. Whole-cell lysates were immunoblotted with primary antibodies against pS-EphA2, EphA2, pRSK, RSK1, RSK2, pY-EGFR, EGFR, β-actin and α-tubulin.

Article Snippet: Antibodies against total EphA2 (C-20; sc-924), RSK1 (C-21; sc-231), RSK2 (C-19; sc-1430), EGFR (1005; sc-03), TAK1 (M-579; sc-7162), β-actin (I-19; sc-1616) and α-tubulin (B-7; sc-5286) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques:

Journal: Nature Communications

Article Title: Crucial roles of RSK in cell motility by catalysing serine phosphorylation of EphA2

doi: 10.1038/ncomms8679

Figure Lengend Snippet: ( a ) A multi-cancer tissue microarray, including 1,010 cores from 13 organ cancer tissues, was adopted for immunohistochemical staining using primary antibodies against pS-EphA2 and pRSK. Typical staining images of lung cancer tissues, including adenocarcinoma (AD) and squamous cell carcinoma (SCC), at low- and high-power magnifications are shown. Scale bar, 20 μm. ( b ) Typical immunohistochemical staining of pS-EphA2 and pRSK in EGFR -mutated (exon 19 deletion) lung adenocarcinoma tissues are shown. Scale bar, 20 μm. ( c – f ) Postoperative overall Kaplan–Meier survival curves of all the lung cancer patients ( c , d ) or smoking patients ( e , f ) were compared according to pRSK negativity or positivity ( c , e ) or pS-EphA2/pRSK double positivity ( d , f ) P values were calculated by the log-rank tests.

Article Snippet: Antibodies against total EphA2 (C-20; sc-924), RSK1 (C-21; sc-231), RSK2 (C-19; sc-1430), EGFR (1005; sc-03), TAK1 (M-579; sc-7162), β-actin (I-19; sc-1616) and α-tubulin (B-7; sc-5286) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Microarray, Immunohistochemical staining, Staining

Journal: PLoS ONE

Article Title: Plasmodium sporozoites can invade hepatocytic cells independently of the Ephrin receptor A2

doi: 10.1371/journal.pone.0200032

Figure Lengend Snippet: A. Hepa1-6 cells were stained with anti-EphA2 D4A2 (blue, left panel) or anti-CD81 MT81 (blue, right panel) followed by Alexa Fluor-488 conjugated secondary antibodies, and analyzed by FACS. The grey histograms represent cells stained with secondary antibody only (negative control). The vertical line represents the threshold used to distinguish EphA2 low and EphA2 high cells B. Hepa1-6 cells were stained with anti-EphA2 D4A2 (rabbit) and anti-CD81 MT81 (rat) primary antibodies, followed by Alexa Fluor-594 (anti-rabbit) and Alexa Fluor-488 (anti-rat) secondary antibodies, and analyzed by FACS (dot plot on the left). As a control, cells were stained with secondary antibodies only (dot plot on the left). There was no correlation between EphA2 and CD81 fluorescence intensities (Pearson coefficient R 2 = 0,263). C. Hepa1-6 cells were transfected with siRNA oligonucleotides targeting mouse EphA2. 48 hours after transfection, surface (left panel) or total (right panel) EphA2 expression was analyzed by FACS in control (blue) and siEphA2-treated (orange) cells. The grey histograms correspond to controls cells stained with secondary antibodies only. D. HepG2 cells were transfected with siRNA oligonucleotides targeting human EphA2. 48 hours after transfection, surface (left panel) or total (right panel) EphA2 expression was analyzed by FACS in control (blue) and siEphA2-treated (orange) cells. The grey histograms correspond to controls cells stained with secondary antibodies only. E. HepG2/CD81 cells were transfected with siRNA oligonucleotides targeting human EphA2 or CD81, or electroporated without siRNA (mock). Cells were lysed 48 hours post-transfection and protein extracts were analyzed by Western blot, using antibodies specific for EphA2 or CD81. F . Hepa1-6 cells were transfected with siRNA oligonucleotides targeting mouse EphA2 or CD81, or with a control siRNA (siCtrl). Cells were lysed 48 hours post-transfection and protein extracts were analyzed by Western blot, using antibodies specific for EphA2 or GAPDH. Among three siRNA oligonucleotides tested against mouse EphA2, siRNA #2 was the most efficient and was used throughout the study.

Article Snippet: Membranes were probed with anti-EphA2 D4A2 XP Rabbit mAb (#6997, Cell Signaling Technology) diluted 1/1000, anti-human CD81 (#HPA007234, Sigma Aldrich) diluted 1/2000, or anti-mouse GADPH (#SC25778, Santa Cruz Biotechnology) diluted 1/5000.

Techniques: Staining, Negative Control, Fluorescence, Transfection, Expressing, Western Blot

Journal: PLoS ONE

Article Title: Plasmodium sporozoites can invade hepatocytic cells independently of the Ephrin receptor A2

doi: 10.1371/journal.pone.0200032

Figure Lengend Snippet: A. Hepa1-6 cells were transfected with siRNA oligonucleotides targeting mouse EphA2 or CD81, or electroporated without siRNA (mock). Cells were infected with PyGFP sporozoites 48 hours after siRNA transfection, and the number of EEFs 24 hours post-infection was determined by fluorescence microscopy after UIS4 staining. NS, non-significant; *, p<0.05 (Kruskal-Wallis with Dunns post-test). B. HepG2/CD81cells were transfected with siRNA oligonucleotides targeting human EphA2 or human CD81, or with a control siRNA (siCtrl). Cells were infected with PyGFP sporozoites 48 hours after siRNA transfection, and the percentage of infected (GFP-positive) cells was determined by FACS 24 hours post-infection. NS, non-significant; *, p<0.05 (Kruskal-Wallis with Dunns post-test). C. HepG2/CD81 cells were transfected with siRNA targeting EphA2 or with a control siRNA (siCtrl) and infected 48 hours later with PyGFP sporozoites. Infected cultures were fixed 24 hours post-infection and stained with anti-UIS4 antibodies (red) and the nuclear stain Hoechst 33342 (blue). The images show PyGFP EEFs (green) surrounded by a UIS4-positive PVM (red). Scale bar, 10 μm. D. Hepa1-6 cells were incubated with PyGFP sporozoites in the presence of increasing concentrations of the EphA2-blocking antibody D4A2, of a control antibody (DA1E, IgG Ctrl, used at the same dilution as D4A2 highest concentration) or without antibody (Ctrl). The number of EEFs was determined 24 hours post-infection by fluorescence microscopy after UIS4 staining. NS, non-significant (Kruskal-Wallis with Dunns post-test).

Article Snippet: Membranes were probed with anti-EphA2 D4A2 XP Rabbit mAb (#6997, Cell Signaling Technology) diluted 1/1000, anti-human CD81 (#HPA007234, Sigma Aldrich) diluted 1/2000, or anti-mouse GADPH (#SC25778, Santa Cruz Biotechnology) diluted 1/5000.

Techniques: Transfection, Infection, Fluorescence, Microscopy, Staining, Incubation, Blocking Assay, Concentration Assay

Journal: PLoS ONE

Article Title: Plasmodium sporozoites can invade hepatocytic cells independently of the Ephrin receptor A2

doi: 10.1371/journal.pone.0200032

Figure Lengend Snippet: A. HepG2 cells were transfected with siRNA oligonucleotides targeting human EphA2 or SR-BI, or with a control siRNA (siCtrl). Cells were infected with PbGFP sporozoites 48 hours after siRNA transfection, and the percentage of infected (GFP-positive) cells was determined by FACS 24 hours post-infection. NS, non-significant; *, p<0.05 (Kruskal-Wallis with Dunns post-test). B. HepG2/CD81 cells were transfected with siRNA oligonucleotides targeting human EphA2 or with a control siRNA (siCtrl). Cells were infected with PbGFP sporozoites 48 hours after siRNA transfection, in the absence or presence of a CD81-blocking antibody (1D6, 20 μg/ml) and the percentage of infected (GFP-positive) cells was determined by FACS 24 hours post-infection. C. Hepa1-6 cells were transfected with siRNA oligonucleotides targeting mouse EphA2 or CD81, or electroporated without siRNA (mock). Cells were infected with PbGFP sporozoites 48 hours after siRNA transfection, and the number of EEFs 24 hours post-infection was determined by fluorescence microscopy after UIS4 staining. NS, non-significant; *, p<0.05 (Kruskal-Wallis with Dunns post-test). D. Hepa1-6 cells were incubated with PbGFP sporozoites in the presence of the EphA2-blocking antibody D4A2 (5 μg/ml) or a control antibody (IgG Ctrl), and the number of EEFs 24 hours post-infection was determined by fluorescence microscopy. NS, non-significant (Mann-Whitney U Test, two-tailed).

Article Snippet: Membranes were probed with anti-EphA2 D4A2 XP Rabbit mAb (#6997, Cell Signaling Technology) diluted 1/1000, anti-human CD81 (#HPA007234, Sigma Aldrich) diluted 1/2000, or anti-mouse GADPH (#SC25778, Santa Cruz Biotechnology) diluted 1/5000.

Techniques: Transfection, Infection, Blocking Assay, Fluorescence, Microscopy, Staining, Incubation, MANN-WHITNEY, Two Tailed Test

Journal: PLoS ONE

Article Title: Plasmodium sporozoites can invade hepatocytic cells independently of the Ephrin receptor A2

doi: 10.1371/journal.pone.0200032

Figure Lengend Snippet: A. Hepa 1–6 cells infected with PbGFP sporozoites were stained 24 hours post-infection with an anti-EphA2 primary antibody (D4A2) followed by Alexa Fluor 594 secondary antibodies (blue histogram), and analyzed by FACS. The grey histogram corresponds to control cells labeled with secondary antibodies only. The vertical line indicates the threshold used to distinguish EphA2 low and EphA2 high cells. B. The percentage of EphA2 low and EphA2 high cells, was determined in non-infected (GFP-negative) and infected (GFP-positive) cells. C. Hepa 1–6 cells infected with PbGFP sporozoites were stained 24 hours post-infection with an anti-CD81 primary antibody (MT81) followed by Alexa Fluor 594 secondary antibodies (blue histogram), and analyzed by FACS. The grey histogram corresponds to control cells labeled with secondary antibodies only. The vertical line indicates the threshold used to distinguish CD81 low and CD81 high cells. This threshold was arbitrarily defined to obtain a majority of CD81 low population after siRNA silencing of CD81. D. The percentage of CD81 low and CD81 high cells, was determined in non-infected (GFP-negative) and infected (GFP-positive) cells. E. Hepa1-6 cells were transfected with siRNA targeting CD81 and infected with PbGFP sporozoites. Cells were stained 24 hours post-infection with an anti-CD81 primary antibody (MT81) followed by Alexa Fluor 594 secondary antibodies (orange histogram), and analyzed by FACS. The grey histogram corresponds to control cells labeled with secondary antibodies only, and the blue histogram corresponds to the control cells labeled with CD81 antibodies, as in C. The vertical line indicates the same threshold as in C, used to distinguish CD81 low and CD81 high cells. F. The percentage of CD81 low and CD81 high cells was determined in non-infected (GFP-negative) and infected (GFP-positive) CD81 siRNA-treated Hepa1-6 cells.

Article Snippet: Membranes were probed with anti-EphA2 D4A2 XP Rabbit mAb (#6997, Cell Signaling Technology) diluted 1/1000, anti-human CD81 (#HPA007234, Sigma Aldrich) diluted 1/2000, or anti-mouse GADPH (#SC25778, Santa Cruz Biotechnology) diluted 1/5000.

Techniques: Infection, Staining, Labeling, Transfection

Journal: PLoS ONE

Article Title: Plasmodium sporozoites can invade hepatocytic cells independently of the Ephrin receptor A2

doi: 10.1371/journal.pone.0200032

Figure Lengend Snippet: A-B . Hepa1-6 cells were transfected with siRNA oligonucleotides targeting mouse EphA2 (siEphA2) or with a control siRNA (siCtrl). 48 hours after siRNA transfection, cells were incubated for 1.5 hours with PyGFP ( A ) or PbGFP ( B ) sporozoites in the presence of rhodamine-labeled dextran, and the number of traversed (dextran-positive) cells was determined by FACS. NS, non-significant (Mann-Whitney U Test, two-tailed).

Article Snippet: Membranes were probed with anti-EphA2 D4A2 XP Rabbit mAb (#6997, Cell Signaling Technology) diluted 1/1000, anti-human CD81 (#HPA007234, Sigma Aldrich) diluted 1/2000, or anti-mouse GADPH (#SC25778, Santa Cruz Biotechnology) diluted 1/5000.

Techniques: Transfection, Incubation, Labeling, MANN-WHITNEY, Two Tailed Test

Journal: bioRxiv

Article Title: EphA7 functions as a receptor for cell-to-cell transmission of Kaposi’s sarcoma-associated herpesvirus into BJAB B cells and for cell-free virus infection by the related rhesus monkey rhadinovirus

doi: 10.1101/522243

Figure Lengend Snippet: Identification of EphA7 as a gH/gL interacting protein in BJAB cells. A) Pulldown from BJAB cells with recombinant soluble gH-FcStrep/gL complexes. The precipitates were separated by PAGE, silver stained and bands at the indicated molecular weight (arrow) were excised and analyzed by mass spectrometry. B) Proteins and number of peptides per protein identified in each sample. C) RNA sequencing Reads Per Kilobase Million (RPKM) of the 14 EPH receptor genes as found in GEO dataset series GSE82184 BJAB-control (accession GSM2185732). D) Pulldown from BJAB and 293T lysate using recombinant soluble KSHV or RRV 26-95 gH/gL complexes as bait. Input amounts were normalized to wet cell pellet. Precipitates were analyzed by Western blot with the indicated antibodies. E) Expression control of EphA2 and EphA7 in BJAB and 293T cells. Lysates were analyzed by Western Blot. The BJAB:293T protein concentration ratio of 2:1 reflects the protein concentration ratio in the pulldown.

Article Snippet: After three washes with lysis buffer, the precipitates were analyzed by polyacrylamide gel electrophoresis and Western blot as described previously ( ) using antibodies to EphA7 (clone E-7, sc-393973) and EphA2 (C-20, sc-924), both from Santa Cruz Biotechnology, 1:100 and 1:500, respectively, in NET gelatine (150mM NaCl, 5mM EDTA 50mM Tris, 0.05% Triton-X-100, 0.25% gelatin, pH 7.5) and donkey anti-mouse HRP-coupled (Dianova) or goat anti-rabbit HRP coupled (Life Technologies) secondary antibody in 5% dry milk powder in PBS with 0.05% Tween20.

Techniques: Recombinant, Staining, Molecular Weight, Mass Spectrometry, RNA Sequencing Assay, Western Blot, Expressing, Protein Concentration

Journal: bioRxiv

Article Title: EphA7 functions as a receptor for cell-to-cell transmission of Kaposi’s sarcoma-associated herpesvirus into BJAB B cells and for cell-free virus infection by the related rhesus monkey rhadinovirus

doi: 10.1101/522243

Figure Lengend Snippet: Knockout of EPHA7 reduces infection by KSHV and RRV. A) Knockout of EPHA7 in BJAB cells. BJAB cells were transduced with the indicated lentiCRISPRv2-based constructs, briefly selected, and cell pools were analyzed by Western blot. B) Absence of detectable EphA2 expression in BJAB cells. BJAB cells were treated as in (A) and analyzed for EphA2 expression by Western blot. One fourth of the protein amount loaded for the BJAB samples was loaded for the 293T EphA2 expression control. C) BJAB cells transduced with the lentiCRISPRv2-based constructs targeting the indicated genes were cocultured with iSLK cells harboring either BAC16 KSHV wt or BAC16 KSHV gH-ELAAN. Infection was analyzed by flow cytometry. The mean across groups of sgRNAs (n=3 infections per sgRNA) targeting EPHA2 (two sgRNAs), EPHA7 (four sgRNAs) or non-targeting (sgNT, two sgRNAs) is indicated by columns. The standard deviation of the mean is indicated by the error bars. The means of the individual triplicate infections for each sgRNA BJAB population within a group are given as symbols within the respective columns. ANOVA and pairwise comparison to the sgNT control group were used to test for significance. D) The same BJAB cell pools as in (C) were infected with cell-free RRV-YFP wt or RRV-YFP gH-AELAAN. Infection was analyzed by flow cytometry.

Article Snippet: After three washes with lysis buffer, the precipitates were analyzed by polyacrylamide gel electrophoresis and Western blot as described previously ( ) using antibodies to EphA7 (clone E-7, sc-393973) and EphA2 (C-20, sc-924), both from Santa Cruz Biotechnology, 1:100 and 1:500, respectively, in NET gelatine (150mM NaCl, 5mM EDTA 50mM Tris, 0.05% Triton-X-100, 0.25% gelatin, pH 7.5) and donkey anti-mouse HRP-coupled (Dianova) or goat anti-rabbit HRP coupled (Life Technologies) secondary antibody in 5% dry milk powder in PBS with 0.05% Tween20.

Techniques: Knock-Out, Infection, Transduction, Construct, Western Blot, Expressing, Flow Cytometry, Standard Deviation

Journal: iScience

Article Title: Lupeol synergizes with 5-fluorouracil to combat c-MET/EphA2 mediated chemoresistance in triple negative breast cancer

doi: 10.1016/j.isci.2023.108395

Figure Lengend Snippet:

Article Snippet: Rabbit Anti human/mouse Phospho-EphA2 (Ser897) (D9A1) , Cell Signaling Technology , Cat#6347; RRID: AB_11220420.

Techniques: Recombinant, Modification, Membrane, Transfection, In Vivo, Software

Journal: Molecular Biology of the Cell

Article Title: Protein kinase A can block EphA2 receptor–mediated cell repulsion by increasing EphA2 S897 phosphorylation

doi: 10.1091/mbc.E16-01-0048

Figure Lengend Snippet: Inhibition of EphA2-dependent cell retraction by representative hit compounds. (A, B) “No compound” refers to cells treated with Fc control or ephrin-A1 Fc and DMSO. Other cells were pretreated for 40 min with the indicated compounds and then for 10 min with Fc or ephrin-A1 Fc in the presence of the compounds. Forskolin was used at 20 μM and dibutyryl cyclical AMP (dbcAMP) at 0.5 mM. The other compounds were used at 5 μM. NECA, 5′-( N -ethylcarboxamido) adenosine. Cells were stained for F-actin with fluorescent phalloidin (red), and nuclei were labeled with DAPI (blue). The inactive compound (O100, oxotremorine methiodide) did not inhibit retraction and rounding of the cells induced by ephrin-A1 Fc, similar to the control. All other compounds shown inhibited retraction, and the cells remained flat. (C) Dose–response curve for forskolin-mediated inhibition of cell retraction analyzed by automated image analysis in the 384-well format. Averages and SEs from quadruplicate measurements from two experiments carried out on different days.

Article Snippet: EphA2 antibodies were from EMD Millipore (05-480 clone D7; Billerica, MA), Thermo Fisher Scientific (34-7400), Santa Cruz Biotechnology (SC-924; Dallas, TX), and R&D Systems (AF3035); antibodies to EphA2 phospho-S897 were from Cell Signaling Technology (6347; Danvers, MA) and Cell Applications (CY1108; San Diego, CA); antibodies to EphA2 phospho-Y588 (12677), CREB phospho-S133 (9196S), CREB (9197S), AKT phospho-S473 (4056S), and AKT (9272S) were from Cell Signaling Technology; the PY20 phosphotyrosine–horseradish peroxidase (HRP) antibody (610012) was from BD Biosciences (Franklin Lakes, NJ); the anti–β-tubulin antibody (T0198) was from Sigma-Aldrich; and the hemagglutinin (HA) antibody (MMS-101R, HA.11 clone 16B12) was from Covance (San Diego, CA).

Techniques: Inhibition, Control, Staining, Labeling

Journal: Molecular Biology of the Cell

Article Title: Protein kinase A can block EphA2 receptor–mediated cell repulsion by increasing EphA2 S897 phosphorylation

doi: 10.1091/mbc.E16-01-0048

Figure Lengend Snippet: Forskolin increases phosphorylation of S897 and other hotspot sites in the EphA2 kinase–SAM domain linker segment. (A) Number of peptides (spectral counts) from the EphA2 kinase–SAM linker segment identified by mass spectrometry as containing the indicated phosphosite in control and forskolin-treated PC3 cells. The sequence of the two peptides containing hotspot residues is shown at the top, with the assigned phosphorylated residues in bold. (B) Left, number of peptides from A with one or two phosphosites, illustrating the dramatic increase in doubly phosphorylated peptides. Right, total number of EphA2 peptides detected, which was similar in control and forskolin-treated cells, indicating that similar total EphA2 levels were analyzed. (C) Number of studies in which each hotspot phosphosite was identified, which provides an indication of the abundance of the phosphosite.

Article Snippet: EphA2 antibodies were from EMD Millipore (05-480 clone D7; Billerica, MA), Thermo Fisher Scientific (34-7400), Santa Cruz Biotechnology (SC-924; Dallas, TX), and R&D Systems (AF3035); antibodies to EphA2 phospho-S897 were from Cell Signaling Technology (6347; Danvers, MA) and Cell Applications (CY1108; San Diego, CA); antibodies to EphA2 phospho-Y588 (12677), CREB phospho-S133 (9196S), CREB (9197S), AKT phospho-S473 (4056S), and AKT (9272S) were from Cell Signaling Technology; the PY20 phosphotyrosine–horseradish peroxidase (HRP) antibody (610012) was from BD Biosciences (Franklin Lakes, NJ); the anti–β-tubulin antibody (T0198) was from Sigma-Aldrich; and the hemagglutinin (HA) antibody (MMS-101R, HA.11 clone 16B12) was from Covance (San Diego, CA).

Techniques: Phospho-proteomics, Mass Spectrometry, Control, Sequencing

Journal: Molecular Biology of the Cell

Article Title: Protein kinase A can block EphA2 receptor–mediated cell repulsion by increasing EphA2 S897 phosphorylation

doi: 10.1091/mbc.E16-01-0048

Figure Lengend Snippet: Forskolin and β2-adrenoceptor agonists increase EphA2 phosphorylation on S897 by PKA as well as phosphorylation on S901. (A) PC3 cells were treated with the PKA inhibitor H89 or the PI3 kinase inhibitor wortmannin (WTM) for 1 h and with forskolin (FSK) for the last 40 min of inhibitor treatment. Cell lysates were probed by immunoblotting with the indicated antibodies. (B) Phosphokinase array signals show the effects of 40-min forskolin treatment on the indicated phosphosites. Images of the duplicate spots on the arrays and exposure times for the autoradiographs are shown at the top. The histogram shows averages from quantification of the spots, with the error bars representing SDs. (C) In vitro kinase reactions with immunoprecipitated EphA2 WT (with and without the H89 PKA inhibitor) or S897A mutant. A control immunoprecipitate is also included. The immunoblot was probed with a phospho-S897–specific antibody and reprobed for EphA2. (D) PC3 cells transfected with empty vector or with constructs encoding PKA WT, the kinase-inactive PKA K72H mutant (PKA KD), or the PKA inhibitor peptide PKI were treated for 30 min with PBS as a control or 10 μM norepinephrine, and lysates were probed with the indicated antibodies. (E) PC3 cells were treated with vehicle DMSO as a control, 20 μM forskolin, or 10 μM indicated β-adrenoceptor agonists for 1 h. Where indicated, cells were treated with 1 μM β-adrenoceptor antagonist propanolol (prop.) for 1 h before the 1-h treatment with β-adrenoceptor agonists. EphA2 immunoprecipitates were probed by immunoblotting for phospho-S897 and reprobed for EphA2. (F) EphA2 WT immunoprecipitated from transiently transfected HEK293 cells was either left untreated or treated with calf-intestinal alkaline phosphatase (CIP) to dephosphorylate the receptor. The control immunoprecipitate was obtained with nonimmune rabbit immunoglobulin Gs. The immunoprecipitates and a cell lysate for comparison were probed with the phospho-S901–specific antibody, a phospho-S897–specific antibody, or an EphA2 antibody. (G) Lysates of HEK293 cells transiently transfected constructs encoding EphA2 WT and the indicated EphA2 mutants or empty vector as a control were probed with the phospho-S901–specific antibody, a phospho-S897–specific antibody, or an EphA2 antibody. (H) In vitro kinase reactions measuring the phosphorylation of immunoprecipitated EphA2 WT and the indicated phosphosite mutants incubated with recombinant CK1 or PKA. EphA2 immunoprecipitates incubated only with ATP without any recombinant kinase served as a control. The EphA2 immunoprecipitates were probed by immunoblotting for phospho-S901 and phospho-S897 and then reprobed for EphA2.

Article Snippet: EphA2 antibodies were from EMD Millipore (05-480 clone D7; Billerica, MA), Thermo Fisher Scientific (34-7400), Santa Cruz Biotechnology (SC-924; Dallas, TX), and R&D Systems (AF3035); antibodies to EphA2 phospho-S897 were from Cell Signaling Technology (6347; Danvers, MA) and Cell Applications (CY1108; San Diego, CA); antibodies to EphA2 phospho-Y588 (12677), CREB phospho-S133 (9196S), CREB (9197S), AKT phospho-S473 (4056S), and AKT (9272S) were from Cell Signaling Technology; the PY20 phosphotyrosine–horseradish peroxidase (HRP) antibody (610012) was from BD Biosciences (Franklin Lakes, NJ); the anti–β-tubulin antibody (T0198) was from Sigma-Aldrich; and the hemagglutinin (HA) antibody (MMS-101R, HA.11 clone 16B12) was from Covance (San Diego, CA).

Techniques: Phospho-proteomics, Western Blot, In Vitro, Immunoprecipitation, Mutagenesis, Control, Transfection, Plasmid Preparation, Construct, Comparison, Incubation, Recombinant

Journal: Molecular Biology of the Cell

Article Title: Protein kinase A can block EphA2 receptor–mediated cell repulsion by increasing EphA2 S897 phosphorylation

doi: 10.1091/mbc.E16-01-0048

Figure Lengend Snippet: PKA activation inhibits EphA2-dependent cell retraction. (A) Representative images of phalloidin-labeled PC3 cells stimulated for 12 min with 0.5 μg/ml control Fc or ephrin-A1 Fc or pretreated for 40 min with 20 μM forskolin or 200 μM of the PKA agonist 6-Benz-cAMP before ephrin-A1 Fc stimulation. Scale bar, 50 μm. (B) Histogram showing average cell areas ± SE under the different conditions (693 cells/condition from three experiments in each of which 77 cells/well from three wells were counted). **** p < 0.0001 for the comparison with the ephrin-A1 Fc condition by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. (C) Cumulative distribution showing the relative frequencies of cells with areas smaller than indicated on the x -axis. (D) Immunoblot of PC3 cells treated with 6-Benz-cAMP or forskolin to assess the levels of EphA2 phosphorylation on S897.

Article Snippet: EphA2 antibodies were from EMD Millipore (05-480 clone D7; Billerica, MA), Thermo Fisher Scientific (34-7400), Santa Cruz Biotechnology (SC-924; Dallas, TX), and R&D Systems (AF3035); antibodies to EphA2 phospho-S897 were from Cell Signaling Technology (6347; Danvers, MA) and Cell Applications (CY1108; San Diego, CA); antibodies to EphA2 phospho-Y588 (12677), CREB phospho-S133 (9196S), CREB (9197S), AKT phospho-S473 (4056S), and AKT (9272S) were from Cell Signaling Technology; the PY20 phosphotyrosine–horseradish peroxidase (HRP) antibody (610012) was from BD Biosciences (Franklin Lakes, NJ); the anti–β-tubulin antibody (T0198) was from Sigma-Aldrich; and the hemagglutinin (HA) antibody (MMS-101R, HA.11 clone 16B12) was from Covance (San Diego, CA).

Techniques: Activation Assay, Labeling, Control, Comparison, Western Blot, Phospho-proteomics

Journal: Molecular Biology of the Cell

Article Title: Protein kinase A can block EphA2 receptor–mediated cell repulsion by increasing EphA2 S897 phosphorylation

doi: 10.1091/mbc.E16-01-0048

Figure Lengend Snippet: Inhibition of EphA2-dependent cell retraction by cAMP requires S897 phosphorylation. (A) Representative images of phalloidin-labeled, EphA2-knockdown PC3 cell populations infected with pLVX-IRES-Neo lentiviral vector (Vector) or lentivirus encoding WT EphA2 (WT) or the indicated EphA2 mutants. The cells, pretreated or not for 40 min with 20 μM forskolin, were stimulated for 12 min with 0.5 μg/ml control Fc or ephrin-A1 Fc. (B) Histogram showing average cell areas ± SE for the different conditions (160 cells/condition from an experiment in which 80 cells/well from two wells were measured). **** p < 0.0001 for the comparison of ephrin-A1 Fc–stimulated cells with the corresponding Fc-stimulated cells by one-way ANOVA followed by Sidak’s multiple comparisons test. (C) Immunoblot of PC3 cells transduced with empty lentiviral vector control and cells expressing the different EphA2 mutants to assess the levels of EphA2 expression and phosphorylation on S897 and S901.

Article Snippet: EphA2 antibodies were from EMD Millipore (05-480 clone D7; Billerica, MA), Thermo Fisher Scientific (34-7400), Santa Cruz Biotechnology (SC-924; Dallas, TX), and R&D Systems (AF3035); antibodies to EphA2 phospho-S897 were from Cell Signaling Technology (6347; Danvers, MA) and Cell Applications (CY1108; San Diego, CA); antibodies to EphA2 phospho-Y588 (12677), CREB phospho-S133 (9196S), CREB (9197S), AKT phospho-S473 (4056S), and AKT (9272S) were from Cell Signaling Technology; the PY20 phosphotyrosine–horseradish peroxidase (HRP) antibody (610012) was from BD Biosciences (Franklin Lakes, NJ); the anti–β-tubulin antibody (T0198) was from Sigma-Aldrich; and the hemagglutinin (HA) antibody (MMS-101R, HA.11 clone 16B12) was from Covance (San Diego, CA).

Techniques: Inhibition, Phospho-proteomics, Labeling, Knockdown, Infection, Plasmid Preparation, Control, Comparison, Western Blot, Transduction, Expressing

Journal: Molecular Biology of the Cell

Article Title: Protein kinase A can block EphA2 receptor–mediated cell repulsion by increasing EphA2 S897 phosphorylation

doi: 10.1091/mbc.E16-01-0048

Figure Lengend Snippet: EphA2 canonical signaling rapidly inhibits AKT but not PKA. (A) PC3 cells stably expressing EphA2 WT or the S897A mutant were treated with 20 μM forskolin for 40 min and/or 0.5 μg/ml ephrin-A1 Fc for 12 min. Lysates were probed by immunoblotting with the indicated antibodies. (B) Quantification of pS897 and pS901 phosphorylation relative to total EphA2 levels and normalized to the value in forskolin-treated cells. Averages ± SE from four independent experiments. The pS897 and pS901 levels in cells stimulated with forskolin and ephrin-A1 Fc are not significantly different from those in cells stimulated only with forskolin by one-sample t test. (C) Normalized phosphokinase array signals show the effects of ephrin-A1 Fc stimulation, with or without forskolin treatment, on the indicated phosphosites. Images of the duplicate spots on the arrays and exposure times for the autoradiographs are shown at the top. The histogram shows averages from quantification of the spots, normalized to the control condition for each phosphosite, with the error bars representing SDs.

Article Snippet: EphA2 antibodies were from EMD Millipore (05-480 clone D7; Billerica, MA), Thermo Fisher Scientific (34-7400), Santa Cruz Biotechnology (SC-924; Dallas, TX), and R&D Systems (AF3035); antibodies to EphA2 phospho-S897 were from Cell Signaling Technology (6347; Danvers, MA) and Cell Applications (CY1108; San Diego, CA); antibodies to EphA2 phospho-Y588 (12677), CREB phospho-S133 (9196S), CREB (9197S), AKT phospho-S473 (4056S), and AKT (9272S) were from Cell Signaling Technology; the PY20 phosphotyrosine–horseradish peroxidase (HRP) antibody (610012) was from BD Biosciences (Franklin Lakes, NJ); the anti–β-tubulin antibody (T0198) was from Sigma-Aldrich; and the hemagglutinin (HA) antibody (MMS-101R, HA.11 clone 16B12) was from Covance (San Diego, CA).

Techniques: Stable Transfection, Expressing, Mutagenesis, Western Blot, Phospho-proteomics, Control

Journal: Molecular Biology of the Cell

Article Title: Protein kinase A can block EphA2 receptor–mediated cell repulsion by increasing EphA2 S897 phosphorylation

doi: 10.1091/mbc.E16-01-0048

Figure Lengend Snippet: PKA phosphorylates EphA2 on S897 in a subset of cancer cells. (A) Prostate cancer and immortalized prostate epithelial cell lines and (B) pancreatic cancer cell lines were treated with vehicle DMSO as a control, 20 μM forskolin, or 10 ng/ml EGF for 20 min. Cell lysates were probed by immunoblotting with the indicated antibodies. pAKT indicates AKT phosphorylated on S473; pCREB indicates CREB phosphorylated on S133. A vertical gap indicates that irrelevant intervening lanes from the blot were removed. Additional control lanes and immunoblots with additional control antibodies are shown in Supplemental Figure S4.

Article Snippet: EphA2 antibodies were from EMD Millipore (05-480 clone D7; Billerica, MA), Thermo Fisher Scientific (34-7400), Santa Cruz Biotechnology (SC-924; Dallas, TX), and R&D Systems (AF3035); antibodies to EphA2 phospho-S897 were from Cell Signaling Technology (6347; Danvers, MA) and Cell Applications (CY1108; San Diego, CA); antibodies to EphA2 phospho-Y588 (12677), CREB phospho-S133 (9196S), CREB (9197S), AKT phospho-S473 (4056S), and AKT (9272S) were from Cell Signaling Technology; the PY20 phosphotyrosine–horseradish peroxidase (HRP) antibody (610012) was from BD Biosciences (Franklin Lakes, NJ); the anti–β-tubulin antibody (T0198) was from Sigma-Aldrich; and the hemagglutinin (HA) antibody (MMS-101R, HA.11 clone 16B12) was from Covance (San Diego, CA).

Techniques: Control, Western Blot