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.

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: 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.

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

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: The phospho-specific antibodies against EphA2 (Ser-897; #6347 and Tyr-588; #12677), Akt (Ser-473; #9271), RSK1 (Ser-380; cross-reacting with RSK2 Ser-386; #11989), ERK (Thr-202/Tyr-204; #9101) and EGFR (Thr-669; #3056, Ser-1046/7; #2238 and Tyr-1068; #2236) were purchased from Cell Signaling Technology (Danvers, MA, 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: The phospho-specific antibodies against EphA2 (Ser-897; #6347 and Tyr-588; #12677), Akt (Ser-473; #9271), RSK1 (Ser-380; cross-reacting with RSK2 Ser-386; #11989), ERK (Thr-202/Tyr-204; #9101) and EGFR (Thr-669; #3056, Ser-1046/7; #2238 and Tyr-1068; #2236) were purchased from Cell Signaling Technology (Danvers, MA, 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: The phospho-specific antibodies against EphA2 (Ser-897; #6347 and Tyr-588; #12677), Akt (Ser-473; #9271), RSK1 (Ser-380; cross-reacting with RSK2 Ser-386; #11989), ERK (Thr-202/Tyr-204; #9101) and EGFR (Thr-669; #3056, Ser-1046/7; #2238 and Tyr-1068; #2236) were purchased from Cell Signaling Technology (Danvers, MA, 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: The phospho-specific antibodies against EphA2 (Ser-897; #6347 and Tyr-588; #12677), Akt (Ser-473; #9271), RSK1 (Ser-380; cross-reacting with RSK2 Ser-386; #11989), ERK (Thr-202/Tyr-204; #9101) and EGFR (Thr-669; #3056, Ser-1046/7; #2238 and Tyr-1068; #2236) were purchased from Cell Signaling Technology (Danvers, MA, 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: The phospho-specific antibodies against EphA2 (Ser-897; #6347 and Tyr-588; #12677), Akt (Ser-473; #9271), RSK1 (Ser-380; cross-reacting with RSK2 Ser-386; #11989), ERK (Thr-202/Tyr-204; #9101) and EGFR (Thr-669; #3056, Ser-1046/7; #2238 and Tyr-1068; #2236) were purchased from Cell Signaling Technology (Danvers, MA, 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: The phospho-specific antibodies against EphA2 (Ser-897; #6347 and Tyr-588; #12677), Akt (Ser-473; #9271), RSK1 (Ser-380; cross-reacting with RSK2 Ser-386; #11989), ERK (Thr-202/Tyr-204; #9101) and EGFR (Thr-669; #3056, Ser-1046/7; #2238 and Tyr-1068; #2236) were purchased from Cell Signaling Technology (Danvers, MA, 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: The phospho-specific antibodies against EphA2 (Ser-897; #6347 and Tyr-588; #12677), Akt (Ser-473; #9271), RSK1 (Ser-380; cross-reacting with RSK2 Ser-386; #11989), ERK (Thr-202/Tyr-204; #9101) and EGFR (Thr-669; #3056, Ser-1046/7; #2238 and Tyr-1068; #2236) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Microarray, Immunohistochemical staining, Staining

Journal: Cells

Article Title: EphA2 Expression in Bone Sarcomas: Bioinformatic Analyses and Preclinical Characterization in Patient-Derived Models of Osteosarcoma, Ewing’s Sarcoma and Chondrosarcoma

doi: 10.3390/cells10112893

Figure Lengend Snippet: Distribution of EphA2 gene expression levels in the Cancer Cell Line Encyclopedia (CCLE). The box plot shows the Z-score-normalized EPHA2 levels in different cell lines divided by cancer type.

Article Snippet: Next, the membranes were incubated overnight at 4 °C with primary antibodies: Phospho-EphA2 (Ser897) (D9A1) Rabbit mAb #6347, EphA2 (D4A2) XP ® Rabbit mAb#6997 and β-Actin (13E5) Rabbit mAb #4970 from Cell Signalling Technologies (Euroclone, Pecco, Italy).

Techniques: Gene Expression

Journal: Cells

Article Title: EphA2 Expression in Bone Sarcomas: Bioinformatic Analyses and Preclinical Characterization in Patient-Derived Models of Osteosarcoma, Ewing’s Sarcoma and Chondrosarcoma

doi: 10.3390/cells10112893

Figure Lengend Snippet: Bioinformatic analysis of EphA2 expression levels from public gene expression datasets on osteosarcoma samples, in relation to patient death status (sample sizes: Yes = 10, 23; No = 27, 30, for GSE39055, GSE21257 datasets, respectively), Huvos grade (sample sizes: G1 = 13, G2 = 16, G3 = 13, G4 = 5; GSE21257 dataset), disease recurrence (sample sizes: Yes = 18, No = 19; GSE39055 dataset), and sex (sample sizes: Male = 20, 34, 23; Female = 17, 19, 30, for GSE39055, GSE21257 and TARGET-OS dataset, respectively). The size of the dots is proportional to the significance of the statistical tests, while the color scale represents the log 2 Fold Change (FC) of expression between the compared groups. Dots with a black border represent tests associated with a p -value lower than 0.05.

Article Snippet: Next, the membranes were incubated overnight at 4 °C with primary antibodies: Phospho-EphA2 (Ser897) (D9A1) Rabbit mAb #6347, EphA2 (D4A2) XP ® Rabbit mAb#6997 and β-Actin (13E5) Rabbit mAb #4970 from Cell Signalling Technologies (Euroclone, Pecco, Italy).

Techniques: Expressing, Gene Expression

Journal: Cells

Article Title: EphA2 Expression in Bone Sarcomas: Bioinformatic Analyses and Preclinical Characterization in Patient-Derived Models of Osteosarcoma, Ewing’s Sarcoma and Chondrosarcoma

doi: 10.3390/cells10112893

Figure Lengend Snippet: Correlation of EphA2 expression level (Log2FC) with patient gender, Huvos grade, outcome and recurrence in osteosarcoma.

Article Snippet: Next, the membranes were incubated overnight at 4 °C with primary antibodies: Phospho-EphA2 (Ser897) (D9A1) Rabbit mAb #6347, EphA2 (D4A2) XP ® Rabbit mAb#6997 and β-Actin (13E5) Rabbit mAb #4970 from Cell Signalling Technologies (Euroclone, Pecco, Italy).

Techniques: Expressing

Journal: Cells

Article Title: EphA2 Expression in Bone Sarcomas: Bioinformatic Analyses and Preclinical Characterization in Patient-Derived Models of Osteosarcoma, Ewing’s Sarcoma and Chondrosarcoma

doi: 10.3390/cells10112893

Figure Lengend Snippet: Bioinformatic analysis of EphA2 expression levels from public gene expression datasets on Ewing’s sarcoma samples, in relation to patient death status (sample sizes: Yes = 11, 14, 27; No = 28, 32 16, for GSE63156, GSE63155 and GSE17674 datasets, respectively), disease recurrence (sample sizes: Yes = 16, 18, No = 23, 28, for GSE63156 and GSE63155 datasets), sex (sample sizes: Male = 20, 27, 66, 28; Female = 19, 19, 51, 16, for GSE63156, GSE63155, GSE34620 and GSE17674 datasets, respectively) and tissue (sample sizes: Tumor= 44; Normal= 19, for GSE17674 dataset). The size of the dots is proportional to the significance of the statistical tests, while the color scale represents the log2 Fold Change (FC) of expression between the compared groups. Dots with a black border represent tests associated with a p -value lower than 0.05.

Article Snippet: Next, the membranes were incubated overnight at 4 °C with primary antibodies: Phospho-EphA2 (Ser897) (D9A1) Rabbit mAb #6347, EphA2 (D4A2) XP ® Rabbit mAb#6997 and β-Actin (13E5) Rabbit mAb #4970 from Cell Signalling Technologies (Euroclone, Pecco, Italy).

Techniques: Expressing, Gene Expression

Journal: Cells

Article Title: EphA2 Expression in Bone Sarcomas: Bioinformatic Analyses and Preclinical Characterization in Patient-Derived Models of Osteosarcoma, Ewing’s Sarcoma and Chondrosarcoma

doi: 10.3390/cells10112893

Figure Lengend Snippet: EPHA2 expression levels and overall survival (OS) probability in dedifferentiated chondrosarcoma group ( n = 16).

Article Snippet: Next, the membranes were incubated overnight at 4 °C with primary antibodies: Phospho-EphA2 (Ser897) (D9A1) Rabbit mAb #6347, EphA2 (D4A2) XP ® Rabbit mAb#6997 and β-Actin (13E5) Rabbit mAb #4970 from Cell Signalling Technologies (Euroclone, Pecco, Italy).

Techniques: Expressing

Journal: Cells

Article Title: EphA2 Expression in Bone Sarcomas: Bioinformatic Analyses and Preclinical Characterization in Patient-Derived Models of Osteosarcoma, Ewing’s Sarcoma and Chondrosarcoma

doi: 10.3390/cells10112893

Figure Lengend Snippet: Boxplot showing association between chondrosarcoma mutational status and EPHA2 expression across different chondrosarcoma subtypes. The selected subtypes were dedifferentiated chondrosarcomas ( n = 16), G1 chondrosarcomas ( n = 17), G2 chondrosarcomas ( n = 38) and G3 chondrosarcomas ( n = 17) * p < 0.05; ** p < 0.01.

Article Snippet: Next, the membranes were incubated overnight at 4 °C with primary antibodies: Phospho-EphA2 (Ser897) (D9A1) Rabbit mAb #6347, EphA2 (D4A2) XP ® Rabbit mAb#6997 and β-Actin (13E5) Rabbit mAb #4970 from Cell Signalling Technologies (Euroclone, Pecco, Italy).

Techniques: Expressing

Journal: Cells

Article Title: EphA2 Expression in Bone Sarcomas: Bioinformatic Analyses and Preclinical Characterization in Patient-Derived Models of Osteosarcoma, Ewing’s Sarcoma and Chondrosarcoma

doi: 10.3390/cells10112893

Figure Lengend Snippet: EphA2 expression in human bone sarcomas and patient-derived xenografts. ( A ) EphA2 mRNA expression in primary human tumors compared to normal counterparts, as obtained by quantitative real time PCR. ( B ) Western blot analysis of phosphorylated-EphA2 (Ser897), EphA2 and β-actin (housekeeping protein as loading control) as obtained from PDX protein extracts. Osteosarcoma (OS-26, OS-29); Ewing Sarcoma (ES-07,ES-15); Chondrosarcoma (CS-281, CS-347). ( C ) Immunohistochemistry showing EphA2 staining in PDXs.

Article Snippet: Next, the membranes were incubated overnight at 4 °C with primary antibodies: Phospho-EphA2 (Ser897) (D9A1) Rabbit mAb #6347, EphA2 (D4A2) XP ® Rabbit mAb#6997 and β-Actin (13E5) Rabbit mAb #4970 from Cell Signalling Technologies (Euroclone, Pecco, Italy).

Techniques: Expressing, Derivative Assay, Real-time Polymerase Chain Reaction, Western Blot, Control, Immunohistochemistry, Staining

Journal: Cells

Article Title: EphA2 Expression in Bone Sarcomas: Bioinformatic Analyses and Preclinical Characterization in Patient-Derived Models of Osteosarcoma, Ewing’s Sarcoma and Chondrosarcoma

doi: 10.3390/cells10112893

Figure Lengend Snippet: Dose-dependent antitumoral effects of the EphA2 inhibitor ALW II-41-27. ( A ) Cell viability assays and ( B ) cell growth assays after 72 h of treatment with scalar concentration of ALW II-41-27. ( C ) Quantification of the plate surface occupied by viable adherent cells, **** p < 0.0001.

Article Snippet: Next, the membranes were incubated overnight at 4 °C with primary antibodies: Phospho-EphA2 (Ser897) (D9A1) Rabbit mAb #6347, EphA2 (D4A2) XP ® Rabbit mAb#6997 and β-Actin (13E5) Rabbit mAb #4970 from Cell Signalling Technologies (Euroclone, Pecco, Italy).

Techniques: Concentration Assay