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: Oncogene

Article Title: Epha2 is a Critical Oncogene in Melanoma

doi: 10.1038/onc.2011.210

Figure Lengend Snippet: Suppression of EphA2 by sh-RNA is also correlated with an increase in p53 levels (A and B). (C). A375 cells devoid of p53 still undergo subG1 accumulation upon EphA2 silencing.

Article Snippet: Antibodies used in this study were as follows: polyclonal anti-EphA2 (C-20), mouse monoclonal anti-p53 (Santa Cruz Biotechnology, Santa Cruz, CA), goat anti-mouse conjugated horseradish peroxidase and goat anti-rabbit IgG-HRP (Cell Signaling Technologies, Beverly, MA).

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 ) 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 ) 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 ) 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: 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