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Cell Signaling Technology Inc antibodies against total epha2

<t>EphA2</t> phosphorylation at Ser-897 is suppressed by SHP2 inhibitors. ( A ) HeLa cells were pretreated with SHP099 (SHP, 10 µM), TNO155 (TNO, 1 µM), BI-D1870 (BI, 10 µM), or trametinib (Tram, 0.03 µM) for 1–2 h and were then stimulated with EGF (10 ng/mL), TNF-⍺ (20 ng/mL), or TPA (10 ng/mL) for 15–30 min. Whole-cell lysates were immunoblotted with EphA2, pS-EphA2, RSK1, pS-RSK1, p-ERK and β-actin antibodies. The relative quantification of pS-EphA2 shown in ( B ) is expressed as the mean ± SD of three independent experiments. P values were calculated by an ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001. ( C ) HeLa cells were pretreated with TNO155 (TNO, 1 µM) or BI-D1870 (BI, 10 µM) for 1 h and were then stimulated with TNF-⍺ (20 ng/mL) for 15 min. The expression of pS-EphA2 and EphA2 was analyzed by immunofluorescence. Representative images from three independent experiments are shown. ( D ) HeLa cells were transfected with siRNAs against GRB2 or the negative control and were then incubated for 48 h. Cells were stimulated with 20 ng/mL TNF-⍺ for 15 min. Whole-cell lysates were immunoblotted with GRB2, EphA2, pS-EphA2, and β-actin antibodies.

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1) Product Images from "Allosteric SHP2 inhibitors suppress lung cancer cell migration by inhibiting non-canonical activation of EphA2 via the ERK-RSK signaling pathway"

Article Title: Allosteric SHP2 inhibitors suppress lung cancer cell migration by inhibiting non-canonical activation of EphA2 via the ERK-RSK signaling pathway

Journal: Scientific Reports

doi: 10.1038/s41598-025-20770-8

EphA2 phosphorylation at Ser-897 is suppressed by SHP2 inhibitors. ( A ) HeLa cells were pretreated with SHP099 (SHP, 10 µM), TNO155 (TNO, 1 µM), BI-D1870 (BI, 10 µM), or trametinib (Tram, 0.03 µM) for 1–2 h and were then stimulated with EGF (10 ng/mL), TNF-⍺ (20 ng/mL), or TPA (10 ng/mL) for 15–30 min. Whole-cell lysates were immunoblotted with EphA2, pS-EphA2, RSK1, pS-RSK1, p-ERK and β-actin antibodies. The relative quantification of pS-EphA2 shown in ( B ) is expressed as the mean ± SD of three independent experiments. P values were calculated by an ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001. ( C ) HeLa cells were pretreated with TNO155 (TNO, 1 µM) or BI-D1870 (BI, 10 µM) for 1 h and were then stimulated with TNF-⍺ (20 ng/mL) for 15 min. The expression of pS-EphA2 and EphA2 was analyzed by immunofluorescence. Representative images from three independent experiments are shown. ( D ) HeLa cells were transfected with siRNAs against GRB2 or the negative control and were then incubated for 48 h. Cells were stimulated with 20 ng/mL TNF-⍺ for 15 min. Whole-cell lysates were immunoblotted with GRB2, EphA2, pS-EphA2, and β-actin antibodies.

Figure Legend Snippet: EphA2 phosphorylation at Ser-897 is suppressed by SHP2 inhibitors. ( A ) HeLa cells were pretreated with SHP099 (SHP, 10 µM), TNO155 (TNO, 1 µM), BI-D1870 (BI, 10 µM), or trametinib (Tram, 0.03 µM) for 1–2 h and were then stimulated with EGF (10 ng/mL), TNF-⍺ (20 ng/mL), or TPA (10 ng/mL) for 15–30 min. Whole-cell lysates were immunoblotted with EphA2, pS-EphA2, RSK1, pS-RSK1, p-ERK and β-actin antibodies. The relative quantification of pS-EphA2 shown in ( B ) is expressed as the mean ± SD of three independent experiments. P values were calculated by an ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001. ( C ) HeLa cells were pretreated with TNO155 (TNO, 1 µM) or BI-D1870 (BI, 10 µM) for 1 h and were then stimulated with TNF-⍺ (20 ng/mL) for 15 min. The expression of pS-EphA2 and EphA2 was analyzed by immunofluorescence. Representative images from three independent experiments are shown. ( D ) HeLa cells were transfected with siRNAs against GRB2 or the negative control and were then incubated for 48 h. Cells were stimulated with 20 ng/mL TNF-⍺ for 15 min. Whole-cell lysates were immunoblotted with GRB2, EphA2, pS-EphA2, and β-actin antibodies.

Techniques Used: Phospho-proteomics, Quantitative Proteomics, Expressing, Immunofluorescence, Transfection, Negative Control, Incubation

Effects of SHP2 inhibitors on non-canonical EphA2 activation in LUAD cells. ( A ) Human lung adenocarcinoma cells (PC-9, HCC827, A925L, and A549) were treated with SHP099 (SHP, 10 µM), TNO155 (TNO, 1 µM), BI-D1870 (BI, 10 µM), or trametinib (Tram, 0.03 µM) for 1 h and were then stimulated with TNF-⍺ (20 ng/mL) for 15 min. Whole-cell lysates were immunoblotted with EphA2, pS-EphA2, RSK1, pS-RSK1, p-ERK and β-actin antibodies. ( B ) The relative quantification of pS-EphA2 shown in (A) is expressed as the mean ± SD of three independent experiments. P values were calculated by an ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001, n.s., not significant.

Figure Legend Snippet: Effects of SHP2 inhibitors on non-canonical EphA2 activation in LUAD cells. ( A ) Human lung adenocarcinoma cells (PC-9, HCC827, A925L, and A549) were treated with SHP099 (SHP, 10 µM), TNO155 (TNO, 1 µM), BI-D1870 (BI, 10 µM), or trametinib (Tram, 0.03 µM) for 1 h and were then stimulated with TNF-⍺ (20 ng/mL) for 15 min. Whole-cell lysates were immunoblotted with EphA2, pS-EphA2, RSK1, pS-RSK1, p-ERK and β-actin antibodies. ( B ) The relative quantification of pS-EphA2 shown in (A) is expressed as the mean ± SD of three independent experiments. P values were calculated by an ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001, n.s., not significant.

Techniques Used: Activation Assay, Quantitative Proteomics

The SHP2 inhibitor suppresses the migration of lung cancer cells. ( A ) PC-9 and A549 cells were treated with SHP099 (SHP, 10 µM), TNO155 (TNO, 1 µM), BI-D1870 (BI, 10 µM), or trametinib (Tram, 0.03 µM) for 1 h. Whole-cell lysates were immunoblotted with EphA2, pS-EphA2, RSK1, p-RSK1, p-ERK and β-actin antibodies. ( B ) The relative quantification of pS-EphA2/EphA2 shown in (A) is expressed as the mean ± SD of three independent experiments. ( C , D ) A549 cells were transfected with siRNAs targeting EphA2 or the negative control RNA and subsequently subjected to a wound healing assay. A monolayer of cells was scratched mechanically and incubated for 48 h. The EphA2 expression was analyzed by immunoblotting. ( E , F ) The wound healing assay was conducted using PC-9 and A549 cells treated with TNO155 (1–3 µM) or BI-D1870 (10 µM) for 24 and 48 h, respectively. ( G , H ) The wound healing assay was conducted using A549-EphA2-KO cells treated with TNO155 (1–3 µM). ( D , F , H ) Quantification of the wound area was performed by the ImageJ software and expressed as the mean ± SD of three independent experiments. P values were calculated by an ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure Legend Snippet: The SHP2 inhibitor suppresses the migration of lung cancer cells. ( A ) PC-9 and A549 cells were treated with SHP099 (SHP, 10 µM), TNO155 (TNO, 1 µM), BI-D1870 (BI, 10 µM), or trametinib (Tram, 0.03 µM) for 1 h. Whole-cell lysates were immunoblotted with EphA2, pS-EphA2, RSK1, p-RSK1, p-ERK and β-actin antibodies. ( B ) The relative quantification of pS-EphA2/EphA2 shown in (A) is expressed as the mean ± SD of three independent experiments. ( C , D ) A549 cells were transfected with siRNAs targeting EphA2 or the negative control RNA and subsequently subjected to a wound healing assay. A monolayer of cells was scratched mechanically and incubated for 48 h. The EphA2 expression was analyzed by immunoblotting. ( E , F ) The wound healing assay was conducted using PC-9 and A549 cells treated with TNO155 (1–3 µM) or BI-D1870 (10 µM) for 24 and 48 h, respectively. ( G , H ) The wound healing assay was conducted using A549-EphA2-KO cells treated with TNO155 (1–3 µM). ( D , F , H ) Quantification of the wound area was performed by the ImageJ software and expressed as the mean ± SD of three independent experiments. P values were calculated by an ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001.

Techniques Used: Migration, Quantitative Proteomics, Transfection, Negative Control, Wound Healing Assay, Incubation, Expressing, Western Blot, Software

Incubation:

Article Title: EphA2 is an epithelial cell pattern recognition receptor for fungal β-glucans.
Article Snippet: .. After 15 and 90 min, the cells were fixed in 3% paraformaldehyde (wt/vol), blocked with 10% BSA (vol/vol) and incubated with antibodies against total EphA2 (Cell Signaling, Danvers, MA, USA; #6997) followed by an AlexaFluor 568-labelled goat anti-rabbit antibody. ..

Article Title: EphA2 is an epithelial cell pattern recognition receptor for fungal β-glucans
Article Snippet: .. After 15, and 90 min, the cells were fixed in 3% paraformaldehyde (wt/vol), blocked with 10% BSA (vol/vol), and incubated with antibodies against total EphA2 (Cell signaling; #6997) followed by an AlexaFluor 568-labeled goat anti-rabbit antibody. ..

Cell-Signaling:

Image Search Results

Journal: Scientific Reports

Article Title: Allosteric SHP2 inhibitors suppress lung cancer cell migration by inhibiting non-canonical activation of EphA2 via the ERK-RSK signaling pathway

doi: 10.1038/s41598-025-20770-8

Figure Lengend Snippet: EphA2 phosphorylation at Ser-897 is suppressed by SHP2 inhibitors. ( A ) HeLa cells were pretreated with SHP099 (SHP, 10 µM), TNO155 (TNO, 1 µM), BI-D1870 (BI, 10 µM), or trametinib (Tram, 0.03 µM) for 1–2 h and were then stimulated with EGF (10 ng/mL), TNF-⍺ (20 ng/mL), or TPA (10 ng/mL) for 15–30 min. Whole-cell lysates were immunoblotted with EphA2, pS-EphA2, RSK1, pS-RSK1, p-ERK and β-actin antibodies. The relative quantification of pS-EphA2 shown in ( B ) is expressed as the mean ± SD of three independent experiments. P values were calculated by an ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001. ( C ) HeLa cells were pretreated with TNO155 (TNO, 1 µM) or BI-D1870 (BI, 10 µM) for 1 h and were then stimulated with TNF-⍺ (20 ng/mL) for 15 min. The expression of pS-EphA2 and EphA2 was analyzed by immunofluorescence. Representative images from three independent experiments are shown. ( D ) HeLa cells were transfected with siRNAs against GRB2 or the negative control and were then incubated for 48 h. Cells were stimulated with 20 ng/mL TNF-⍺ for 15 min. Whole-cell lysates were immunoblotted with GRB2, EphA2, pS-EphA2, and β-actin antibodies.

Article Snippet: Phospho-specific antibodies against total EphA2 (D4A2; #6997S), phospho-EphA2 (Ser-897; #6347), phospho-ERK (Thr-202/Tyr-204; #9101), phospho-p38 (Thr-180/Tyr-182; #4511S), phospho-JNK (Thr-183/Tyr-185; #4671), phospho-p65 (Ser-536; #3033), phospho-SHP2 (Tyr-542; #15543), and phospho-RSK1 (Ser-380; #11989) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Phospho-proteomics, Quantitative Proteomics, Expressing, Immunofluorescence, Transfection, Negative Control, Incubation

Journal: Scientific Reports

Article Title: Allosteric SHP2 inhibitors suppress lung cancer cell migration by inhibiting non-canonical activation of EphA2 via the ERK-RSK signaling pathway

doi: 10.1038/s41598-025-20770-8

Figure Lengend Snippet: Effects of SHP2 inhibitors on non-canonical EphA2 activation in LUAD cells. ( A ) Human lung adenocarcinoma cells (PC-9, HCC827, A925L, and A549) were treated with SHP099 (SHP, 10 µM), TNO155 (TNO, 1 µM), BI-D1870 (BI, 10 µM), or trametinib (Tram, 0.03 µM) for 1 h and were then stimulated with TNF-⍺ (20 ng/mL) for 15 min. Whole-cell lysates were immunoblotted with EphA2, pS-EphA2, RSK1, pS-RSK1, p-ERK and β-actin antibodies. ( B ) The relative quantification of pS-EphA2 shown in (A) is expressed as the mean ± SD of three independent experiments. P values were calculated by an ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001, n.s., not significant.

Techniques: Activation Assay, Quantitative Proteomics

Journal: Scientific Reports

Article Title: Allosteric SHP2 inhibitors suppress lung cancer cell migration by inhibiting non-canonical activation of EphA2 via the ERK-RSK signaling pathway

doi: 10.1038/s41598-025-20770-8

Figure Lengend Snippet: The SHP2 inhibitor suppresses the migration of lung cancer cells. ( A ) PC-9 and A549 cells were treated with SHP099 (SHP, 10 µM), TNO155 (TNO, 1 µM), BI-D1870 (BI, 10 µM), or trametinib (Tram, 0.03 µM) for 1 h. Whole-cell lysates were immunoblotted with EphA2, pS-EphA2, RSK1, p-RSK1, p-ERK and β-actin antibodies. ( B ) The relative quantification of pS-EphA2/EphA2 shown in (A) is expressed as the mean ± SD of three independent experiments. ( C , D ) A549 cells were transfected with siRNAs targeting EphA2 or the negative control RNA and subsequently subjected to a wound healing assay. A monolayer of cells was scratched mechanically and incubated for 48 h. The EphA2 expression was analyzed by immunoblotting. ( E , F ) The wound healing assay was conducted using PC-9 and A549 cells treated with TNO155 (1–3 µM) or BI-D1870 (10 µM) for 24 and 48 h, respectively. ( G , H ) The wound healing assay was conducted using A549-EphA2-KO cells treated with TNO155 (1–3 µM). ( D , F , H ) Quantification of the wound area was performed by the ImageJ software and expressed as the mean ± SD of three independent experiments. P values were calculated by an ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001.

Techniques: Migration, Quantitative Proteomics, Transfection, Negative Control, Wound Healing Assay, Incubation, Expressing, Western Blot, Software

( 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.

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

( 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.

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.

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

( 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.

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.

Techniques: SDS Page

( 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.

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.

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

( 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.

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.

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

( 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.

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.

Techniques:

( 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.

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.

Techniques: Microarray, Immunohistochemical staining, Staining

Review

antibodies against total epha2 (Cell Signaling Technology Inc)

Structured Review

Images

1) Product Images from "Allosteric SHP2 inhibitors suppress lung cancer cell migration by inhibiting non-canonical activation of EphA2 via the ERK-RSK signaling pathway"

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