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Fig. 2 Progranulin and <t>EphA2</t> differ in their ability to modulate mesothelioma cell motility and adhesion. A Migration of parental, GRN KO and progranulin-re-expressing GRN KO MSTO-211H cells was assessed using transwells as described in Material and Methods. B Migration of parental, GRN KO and EphA2 KO MSTO-211H cells as assessed by transwells migration. C Invasion of parental, GRN KO and EphA2 KO MSTO-211H cells was assessed using matrigel-coated transwells. D The ability of parental, GRN KO and EphA2 KO MSTO-211H cells to adhere to plasma fibronectin, collagen and poly-L-Lys was assessed as described in Material and Methods. E The migratory ability of parental and progranulin overexpressing NCI-H2052 cells was assessed using transwells. F Transwell migration of parental and EphA2 KO NCI-H2052 cells, untreated or stimulated with 50 nM progranulin. G Invasion through matrigel-coated transwells of parental and EphA2 KO NCI-H2052 cells, untreated or stimulated with 50 nM progranulin. For motility and adhesion assays, data are the average of three independent experiments ± SD. *** p < 0.001, ** p < 0.01, * p < 0.05. H Parental, GRN KO and EphA2 KO MSTO-211H cells were subcutaneously implanted in Rag2-/- mice and tumor volumes measured at the indicated time post tumor injection. N = 8, ± SD, * p < 0.05, ** p < 0.01

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1) Product Images from "Complexity of progranulin mechanisms of action in mesothelioma."

Article Title: Complexity of progranulin mechanisms of action in mesothelioma.

Journal: Journal of experimental & clinical cancer research : CR

doi: 10.1186/s13046-022-02546-4

Fig. 2 Progranulin and EphA2 differ in their ability to modulate mesothelioma cell motility and adhesion. A Migration of parental, GRN KO and progranulin-re-expressing GRN KO MSTO-211H cells was assessed using transwells as described in Material and Methods. B Migration of parental, GRN KO and EphA2 KO MSTO-211H cells as assessed by transwells migration. C Invasion of parental, GRN KO and EphA2 KO MSTO-211H cells was assessed using matrigel-coated transwells. D The ability of parental, GRN KO and EphA2 KO MSTO-211H cells to adhere to plasma fibronectin, collagen and poly-L-Lys was assessed as described in Material and Methods. E The migratory ability of parental and progranulin overexpressing NCI-H2052 cells was assessed using transwells. F Transwell migration of parental and EphA2 KO NCI-H2052 cells, untreated or stimulated with 50 nM progranulin. G Invasion through matrigel-coated transwells of parental and EphA2 KO NCI-H2052 cells, untreated or stimulated with 50 nM progranulin. For motility and adhesion assays, data are the average of three independent experiments ± SD. *** p < 0.001, ** p < 0.01, * p < 0.05. H Parental, GRN KO and EphA2 KO MSTO-211H cells were subcutaneously implanted in Rag2-/- mice and tumor volumes measured at the indicated time post tumor injection. N = 8, ± SD, * p < 0.05, ** p < 0.01

Figure Legend Snippet: Fig. 2 Progranulin and EphA2 differ in their ability to modulate mesothelioma cell motility and adhesion. A Migration of parental, GRN KO and progranulin-re-expressing GRN KO MSTO-211H cells was assessed using transwells as described in Material and Methods. B Migration of parental, GRN KO and EphA2 KO MSTO-211H cells as assessed by transwells migration. C Invasion of parental, GRN KO and EphA2 KO MSTO-211H cells was assessed using matrigel-coated transwells. D The ability of parental, GRN KO and EphA2 KO MSTO-211H cells to adhere to plasma fibronectin, collagen and poly-L-Lys was assessed as described in Material and Methods. E The migratory ability of parental and progranulin overexpressing NCI-H2052 cells was assessed using transwells. F Transwell migration of parental and EphA2 KO NCI-H2052 cells, untreated or stimulated with 50 nM progranulin. G Invasion through matrigel-coated transwells of parental and EphA2 KO NCI-H2052 cells, untreated or stimulated with 50 nM progranulin. For motility and adhesion assays, data are the average of three independent experiments ± SD. *** p < 0.001, ** p < 0.01, * p < 0.05. H Parental, GRN KO and EphA2 KO MSTO-211H cells were subcutaneously implanted in Rag2-/- mice and tumor volumes measured at the indicated time post tumor injection. N = 8, ± SD, * p < 0.05, ** p < 0.01

Techniques Used: Migration, Expressing, Clinical Proteomics, Injection

Fig. 4 FAK mediates progranulin-dependent activation of AKT and ERK1/2 in mesothelioma cells. A, B Parental and EphA2 KO MSTO-211H (A) and NCI-H2052 (B) cells were serum-starved for 24 h, pre-incubated with the FAK inhibitor PF-573228 (5 µM) for 1 h and then treated with the same concentration of PF-573228 alone or in combination with 50 nM progranulin for 15 min. Levels of total and phosphorylated EphA2 (S897), AKT, ERK1/2 and FAK (Y397) were assessed by immunoblot. C NCI-H2052 cells were transfected with siRNA targeting FAK (PTK2) or non-targeting control (siCTR) siRNA. At 48 h post-transfection cells were transferred onto serum-free medium and 24 h later exposed to 50 nM progranulin for 15 min. Levels of total and phosphorylated EphA2 (S897), AKT, ERK1/2 and of FAK as assessed by immunoblot

Figure Legend Snippet: Fig. 4 FAK mediates progranulin-dependent activation of AKT and ERK1/2 in mesothelioma cells. A, B Parental and EphA2 KO MSTO-211H (A) and NCI-H2052 (B) cells were serum-starved for 24 h, pre-incubated with the FAK inhibitor PF-573228 (5 µM) for 1 h and then treated with the same concentration of PF-573228 alone or in combination with 50 nM progranulin for 15 min. Levels of total and phosphorylated EphA2 (S897), AKT, ERK1/2 and FAK (Y397) were assessed by immunoblot. C NCI-H2052 cells were transfected with siRNA targeting FAK (PTK2) or non-targeting control (siCTR) siRNA. At 48 h post-transfection cells were transferred onto serum-free medium and 24 h later exposed to 50 nM progranulin for 15 min. Levels of total and phosphorylated EphA2 (S897), AKT, ERK1/2 and of FAK as assessed by immunoblot

Techniques Used: Activation Assay, Incubation, Concentration Assay, Western Blot, Transfection, Control

Fig. 7 Progranulin modulates pFAK Y397 levels in a RYK-dependent manner and modulates focal adhesion turn over. A Parental (P), EphA2 KO and EphA2 KO MSTO-211H cells stably transfected with an empty vector, re-expressing wild type EphA2, or EphA2 mutants were transfected with siRNAs targeting progranulin (siGRN) or non-targeting (siCTR) control. At 8 h post-transfection cells were serum-starved and total and phosphorylated FAK (Y397) were analyzed by immunoblot 48 h post-transfection. B Phosphorylated FAK (Y397) levels in parental, GRN KO, GRN KO MSTO-211H cells with reconstituted progranulin expression (left panel) or parental and PGRN overexpressing NCI-H2052 cells (right panel) serum-starved for 24 h. C Phosphorylated FAK (Y397) in siRYK- or siControl-transfected (siCTR) MSTO-211H and NCI-H2052 cells serum-starved for 24 h, pre-incubated with gefitinib (10 µM) for 1 h and then treated with gefitinib alone or in combination with 50 nM progranulin for 15 min. D Levels of total and phosphorylated FAK (Y397) in EphA2 KO MSTO-211H cells transfected with siRNA targeting RYK or control (siCTR) siRNA, serum-starved for 24 h and stimulated with 50 nM progranulin for 15 min. E Levels of total and phosphorylated FAK (Y397) in MSTO-211H cells transfected with either siRNAs targeting progranulin, RYK, their combination or non-targeting controls (siCTR) and serum-starved for 40 h. F Levels of total and phosphorylated FAK (Y397) in parental and GRN KO MSTO-211H cells transfected with siRYK or control (siCTR) siRNA and serum-starved for 24 h. G-I Parental and GRN KO MSTO-211H cells (G), parental MSTO-211H cells 24 h post transfection with siRNA targeting progranulin (siGRN) or control ( siCTR) siRNA (H), and GRN KO MSTO-211H cells 48 h post transfection with RYK-specific siRNA or control (siCTR) siRNA (I) were serum-starved for 24 h and then treated with 10 µM nocodazole (NCZ) for 4 h. Nocodazole was then washed-out and the levels of phosphorylated FAK (Y397) were analyzed by immunoblot in cell lysates at the indicated times after nocodazole washout

Figure Legend Snippet: Fig. 7 Progranulin modulates pFAK Y397 levels in a RYK-dependent manner and modulates focal adhesion turn over. A Parental (P), EphA2 KO and EphA2 KO MSTO-211H cells stably transfected with an empty vector, re-expressing wild type EphA2, or EphA2 mutants were transfected with siRNAs targeting progranulin (siGRN) or non-targeting (siCTR) control. At 8 h post-transfection cells were serum-starved and total and phosphorylated FAK (Y397) were analyzed by immunoblot 48 h post-transfection. B Phosphorylated FAK (Y397) levels in parental, GRN KO, GRN KO MSTO-211H cells with reconstituted progranulin expression (left panel) or parental and PGRN overexpressing NCI-H2052 cells (right panel) serum-starved for 24 h. C Phosphorylated FAK (Y397) in siRYK- or siControl-transfected (siCTR) MSTO-211H and NCI-H2052 cells serum-starved for 24 h, pre-incubated with gefitinib (10 µM) for 1 h and then treated with gefitinib alone or in combination with 50 nM progranulin for 15 min. D Levels of total and phosphorylated FAK (Y397) in EphA2 KO MSTO-211H cells transfected with siRNA targeting RYK or control (siCTR) siRNA, serum-starved for 24 h and stimulated with 50 nM progranulin for 15 min. E Levels of total and phosphorylated FAK (Y397) in MSTO-211H cells transfected with either siRNAs targeting progranulin, RYK, their combination or non-targeting controls (siCTR) and serum-starved for 40 h. F Levels of total and phosphorylated FAK (Y397) in parental and GRN KO MSTO-211H cells transfected with siRYK or control (siCTR) siRNA and serum-starved for 24 h. G-I Parental and GRN KO MSTO-211H cells (G), parental MSTO-211H cells 24 h post transfection with siRNA targeting progranulin (siGRN) or control ( siCTR) siRNA (H), and GRN KO MSTO-211H cells 48 h post transfection with RYK-specific siRNA or control (siCTR) siRNA (I) were serum-starved for 24 h and then treated with 10 µM nocodazole (NCZ) for 4 h. Nocodazole was then washed-out and the levels of phosphorylated FAK (Y397) were analyzed by immunoblot in cell lysates at the indicated times after nocodazole washout

Techniques Used: Stable Transfection, Transfection, Plasmid Preparation, Expressing, Control, Western Blot, Incubation

Image Search Results

FIGURE 4 Analysis of S1-mediated crystal lattice packing interactions in FabS1CE-C1:-EPHA2-CRD and FabS1C-C1 structures. (a) Crystal lattice packing arrangement (upper panel) with symmetry mates, and asymmetric unit (lower panel) of (i) FabS1CE-C1: EPHA2-CRD (P3221 space group, trigonal crystal system), (ii) FabS1CE-C1:EPHA2-CRD (P21 space group, monoclinic crystal system), and (iii) FabS1C-C1 (P21 space group). ASU Fab heavy- and light-chains are colored light blue or gray, respectively. Symmetry mate Fab heavy- and light-chains are colored dark blue and green, respectively. EPHA2-CRD is colored magenta. The heavy- and light-chain variable (VH and VL) and constant domains (CH and CL) of the Fab are labeled in (i). (b) S1 substitutions (Q165S and K167Y) and residues in the nearby loop region (N170, A171, L172. S174) cooperate to form crystal lattice packing sites in the following structures: (i) FabS1CE-C1: EPHA2-CRD (P3221 space group), (ii) FabS1CE-C1:EPHA2-CRD (P21 space group), and (iii and iv) FabS1C-C1. NB: In the S1-crystal lattice packing site of FabS1CE-C1:EPHA2-CRD (P21 space group) (ii), the K72 side chain of the packing Fab VH domain remains partially unresolved from the electron density indicating a dynamic interaction.

Journal: Protein science : a publication of the Protein Society

Article Title: Engineered antigen-binding fragments for enhanced crystallization of antibody:antigen complexes.

doi: 10.1002/pro.4824

Figure Lengend Snippet: FIGURE 4 Analysis of S1-mediated crystal lattice packing interactions in FabS1CE-C1:-EPHA2-CRD and FabS1C-C1 structures. (a) Crystal lattice packing arrangement (upper panel) with symmetry mates, and asymmetric unit (lower panel) of (i) FabS1CE-C1: EPHA2-CRD (P3221 space group, trigonal crystal system), (ii) FabS1CE-C1:EPHA2-CRD (P21 space group, monoclinic crystal system), and (iii) FabS1C-C1 (P21 space group). ASU Fab heavy- and light-chains are colored light blue or gray, respectively. Symmetry mate Fab heavy- and light-chains are colored dark blue and green, respectively. EPHA2-CRD is colored magenta. The heavy- and light-chain variable (VH and VL) and constant domains (CH and CL) of the Fab are labeled in (i). (b) S1 substitutions (Q165S and K167Y) and residues in the nearby loop region (N170, A171, L172. S174) cooperate to form crystal lattice packing sites in the following structures: (i) FabS1CE-C1: EPHA2-CRD (P3221 space group), (ii) FabS1CE-C1:EPHA2-CRD (P21 space group), and (iii and iv) FabS1C-C1. NB: In the S1-crystal lattice packing site of FabS1CE-C1:EPHA2-CRD (P21 space group) (ii), the K72 side chain of the packing Fab VH domain remains partially unresolved from the electron density indicating a dynamic interaction.

Article Snippet: Cell culture expressing EPHA2-CRD or antigen-B was supplemented with 5 mM Kifunensine (MedChemExpress) to inhibit mannosidase I activity.

Techniques: Labeling

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Complexity of progranulin mechanisms of action in mesothelioma.

doi: 10.1186/s13046-022-02546-4

Figure Lengend Snippet: Fig. 2 Progranulin and EphA2 differ in their ability to modulate mesothelioma cell motility and adhesion. A Migration of parental, GRN KO and progranulin-re-expressing GRN KO MSTO-211H cells was assessed using transwells as described in Material and Methods. B Migration of parental, GRN KO and EphA2 KO MSTO-211H cells as assessed by transwells migration. C Invasion of parental, GRN KO and EphA2 KO MSTO-211H cells was assessed using matrigel-coated transwells. D The ability of parental, GRN KO and EphA2 KO MSTO-211H cells to adhere to plasma fibronectin, collagen and poly-L-Lys was assessed as described in Material and Methods. E The migratory ability of parental and progranulin overexpressing NCI-H2052 cells was assessed using transwells. F Transwell migration of parental and EphA2 KO NCI-H2052 cells, untreated or stimulated with 50 nM progranulin. G Invasion through matrigel-coated transwells of parental and EphA2 KO NCI-H2052 cells, untreated or stimulated with 50 nM progranulin. For motility and adhesion assays, data are the average of three independent experiments ± SD. *** p < 0.001, ** p < 0.01, * p < 0.05. H Parental, GRN KO and EphA2 KO MSTO-211H cells were subcutaneously implanted in Rag2-/- mice and tumor volumes measured at the indicated time post tumor injection. N = 8, ± SD, * p < 0.05, ** p < 0.01

Article Snippet: To reconstitute EphA2 expression in EphA2 KO MSTO-211H cells, we used the retroviral plasmid pCLXSN-EphA2-Flag, a gift from Jin Chen (Addgene plasmid #102755, Addgene, Wartertown, MA, USA).

Techniques: Migration, Expressing, Clinical Proteomics, Injection

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Complexity of progranulin mechanisms of action in mesothelioma.

doi: 10.1186/s13046-022-02546-4

Figure Lengend Snippet: Fig. 4 FAK mediates progranulin-dependent activation of AKT and ERK1/2 in mesothelioma cells. A, B Parental and EphA2 KO MSTO-211H (A) and NCI-H2052 (B) cells were serum-starved for 24 h, pre-incubated with the FAK inhibitor PF-573228 (5 µM) for 1 h and then treated with the same concentration of PF-573228 alone or in combination with 50 nM progranulin for 15 min. Levels of total and phosphorylated EphA2 (S897), AKT, ERK1/2 and FAK (Y397) were assessed by immunoblot. C NCI-H2052 cells were transfected with siRNA targeting FAK (PTK2) or non-targeting control (siCTR) siRNA. At 48 h post-transfection cells were transferred onto serum-free medium and 24 h later exposed to 50 nM progranulin for 15 min. Levels of total and phosphorylated EphA2 (S897), AKT, ERK1/2 and of FAK as assessed by immunoblot

Techniques: Activation Assay, Incubation, Concentration Assay, Western Blot, Transfection, Control

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Complexity of progranulin mechanisms of action in mesothelioma.

doi: 10.1186/s13046-022-02546-4

Figure Lengend Snippet: Fig. 7 Progranulin modulates pFAK Y397 levels in a RYK-dependent manner and modulates focal adhesion turn over. A Parental (P), EphA2 KO and EphA2 KO MSTO-211H cells stably transfected with an empty vector, re-expressing wild type EphA2, or EphA2 mutants were transfected with siRNAs targeting progranulin (siGRN) or non-targeting (siCTR) control. At 8 h post-transfection cells were serum-starved and total and phosphorylated FAK (Y397) were analyzed by immunoblot 48 h post-transfection. B Phosphorylated FAK (Y397) levels in parental, GRN KO, GRN KO MSTO-211H cells with reconstituted progranulin expression (left panel) or parental and PGRN overexpressing NCI-H2052 cells (right panel) serum-starved for 24 h. C Phosphorylated FAK (Y397) in siRYK- or siControl-transfected (siCTR) MSTO-211H and NCI-H2052 cells serum-starved for 24 h, pre-incubated with gefitinib (10 µM) for 1 h and then treated with gefitinib alone or in combination with 50 nM progranulin for 15 min. D Levels of total and phosphorylated FAK (Y397) in EphA2 KO MSTO-211H cells transfected with siRNA targeting RYK or control (siCTR) siRNA, serum-starved for 24 h and stimulated with 50 nM progranulin for 15 min. E Levels of total and phosphorylated FAK (Y397) in MSTO-211H cells transfected with either siRNAs targeting progranulin, RYK, their combination or non-targeting controls (siCTR) and serum-starved for 40 h. F Levels of total and phosphorylated FAK (Y397) in parental and GRN KO MSTO-211H cells transfected with siRYK or control (siCTR) siRNA and serum-starved for 24 h. G-I Parental and GRN KO MSTO-211H cells (G), parental MSTO-211H cells 24 h post transfection with siRNA targeting progranulin (siGRN) or control ( siCTR) siRNA (H), and GRN KO MSTO-211H cells 48 h post transfection with RYK-specific siRNA or control (siCTR) siRNA (I) were serum-starved for 24 h and then treated with 10 µM nocodazole (NCZ) for 4 h. Nocodazole was then washed-out and the levels of phosphorylated FAK (Y397) were analyzed by immunoblot in cell lysates at the indicated times after nocodazole washout

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Expressing, Control, Western Blot, Incubation

Progranulin, Ephrin-A1, and EphA2 expression in bladder cancer. (A) mRNA levels of GRN and EFNA1 in normal or urothelial carcinomas of low and high grade (Oncomine). (B) Steady state levels of progranulin (GRN) and ephrin-A1 (EFNA1) in T24 and UMUC-3 cells; mean ±SEM, n = 3. (C) Micrographs from the human protein Atlas depicting representative images of normal bladder and low or high-grade urothelial carcinomas. (D) EphA2 expression levels in bladder cancer tissues using IHC on a bladder cancer TMA. (E) Quantification of EphA2 expression in tissues using ImageJ; bladder, n = 27; T1-T4 urothelial carcinomas, n = 123; ***p<0.001. (F) EphA2 expression in various urothelial cancer cells by immunoblot using anti-EphA2 and anti-β-actin antibodies.

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Progranulin/EphA2 axis: A novel oncogenic mechanism in bladder cancer

doi: 10.1016/j.matbio.2020.03.009

Figure Lengend Snippet: Progranulin, Ephrin-A1, and EphA2 expression in bladder cancer. (A) mRNA levels of GRN and EFNA1 in normal or urothelial carcinomas of low and high grade (Oncomine). (B) Steady state levels of progranulin (GRN) and ephrin-A1 (EFNA1) in T24 and UMUC-3 cells; mean ±SEM, n = 3. (C) Micrographs from the human protein Atlas depicting representative images of normal bladder and low or high-grade urothelial carcinomas. (D) EphA2 expression levels in bladder cancer tissues using IHC on a bladder cancer TMA. (E) Quantification of EphA2 expression in tissues using ImageJ; bladder, n = 27; T1-T4 urothelial carcinomas, n = 123; ***p<0.001. (F) EphA2 expression in various urothelial cancer cells by immunoblot using anti-EphA2 and anti-β-actin antibodies.

Article Snippet: Generation of EphA2-depleted bladder cancer cells Cell lines (UMUC-3) stably depleted of endogenous EphA2 were generated by transfecting the pRS-shScr (scrambled shRNAs) and pRS/shEphA2 plasmids expressing shRNAs against human EphA2 (OriGene Technologies, Inc.) using the TransIT ® -Prostate Transfection Kit (Mirus).

Techniques: Expressing, Western Blot

Progranulin evokes EphA2 phosphorylation. Serum-starved T24 cells were exposed to progranulin (150 nM, 15 min) and immunoprecipitated with anti-EphA2 antibodies. Coomassie-stained bands of interest were trypsin-digested and analyzed by LC-MS/MS on a Q Exactive™ Plus mass spec. The mass spec data were probed against the UniProt human database for STY phosphorylation. False discovery rate for peptides/site identifications was set at 1%. (A) Modifications of various residues shown as the sum of MS peptides intensities. (B) EphA2 schematic showing the location of phosphorylated residues. (C-D) Western immunoblots of serum-starved T24 cells exposed to 150 nM progranulin using several Phospho-specific and total antibodies. (E) Western immunoblots of serum-starved 5637 cells after progranulin stimulation ±specific inhibitors for PI3K pathway (LY294002, 20 μM) or Erk1/2 (U0126, 10 μM). (F) Phospho-EphA2 (Ser897) assessed by immunoblot in UMUC-3/shScr (control) and UMUC-3/shPGRN cells. (G) Inhibition of EphA2 kinase activity blocks progranulin-induced phosphorylation. Western immunoblots of serum-starved T24 cells exposed to Ephrin-A1-Fc (2.1 nM) or progranulin (150 nM) for 15 min ±ALW-II-41–27 (1 μM), and probed with Phospho-EphA2 (Ser897) and EphA2 antibodies.

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Progranulin/EphA2 axis: A novel oncogenic mechanism in bladder cancer

doi: 10.1016/j.matbio.2020.03.009

Figure Lengend Snippet: Progranulin evokes EphA2 phosphorylation. Serum-starved T24 cells were exposed to progranulin (150 nM, 15 min) and immunoprecipitated with anti-EphA2 antibodies. Coomassie-stained bands of interest were trypsin-digested and analyzed by LC-MS/MS on a Q Exactive™ Plus mass spec. The mass spec data were probed against the UniProt human database for STY phosphorylation. False discovery rate for peptides/site identifications was set at 1%. (A) Modifications of various residues shown as the sum of MS peptides intensities. (B) EphA2 schematic showing the location of phosphorylated residues. (C-D) Western immunoblots of serum-starved T24 cells exposed to 150 nM progranulin using several Phospho-specific and total antibodies. (E) Western immunoblots of serum-starved 5637 cells after progranulin stimulation ±specific inhibitors for PI3K pathway (LY294002, 20 μM) or Erk1/2 (U0126, 10 μM). (F) Phospho-EphA2 (Ser897) assessed by immunoblot in UMUC-3/shScr (control) and UMUC-3/shPGRN cells. (G) Inhibition of EphA2 kinase activity blocks progranulin-induced phosphorylation. Western immunoblots of serum-starved T24 cells exposed to Ephrin-A1-Fc (2.1 nM) or progranulin (150 nM) for 15 min ±ALW-II-41–27 (1 μM), and probed with Phospho-EphA2 (Ser897) and EphA2 antibodies.

Techniques: Immunoprecipitation, Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Western Blot, Inhibition, Activity Assay

EphA2 is required for progranulin-evoked activity and cisplatin sensitivity. (A) EphA2 was depleted in UMUC-3 cells by siRNA approaches and EphA2 expression levels were assessed by immunoblot with anti-EphA2 polyclonal antibodies and normalized over β-actin content. Densitometric analysis was performed using ImageJ (National Institutes of Health) and expressed as arbitrary units. (B) Motility assays in Boyden chambers ±progranulin, ***p<0.001. (C) Anchorage-independent growth in soft-agar was performed as described in Materials and Methods ***p<0.001. (D) Cell survival as assessed by a Colorimetric Cell Cytotoxicity Assay Kit at various cisplatin concentrations. Mean ±SD; n = 3 biological replicates run in duplicates. **p<0.005; ***p<0.001.

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Progranulin/EphA2 axis: A novel oncogenic mechanism in bladder cancer

doi: 10.1016/j.matbio.2020.03.009

Figure Lengend Snippet: EphA2 is required for progranulin-evoked activity and cisplatin sensitivity. (A) EphA2 was depleted in UMUC-3 cells by siRNA approaches and EphA2 expression levels were assessed by immunoblot with anti-EphA2 polyclonal antibodies and normalized over β-actin content. Densitometric analysis was performed using ImageJ (National Institutes of Health) and expressed as arbitrary units. (B) Motility assays in Boyden chambers ±progranulin, ***p<0.001. (C) Anchorage-independent growth in soft-agar was performed as described in Materials and Methods ***p<0.001. (D) Cell survival as assessed by a Colorimetric Cell Cytotoxicity Assay Kit at various cisplatin concentrations. Mean ±SD; n = 3 biological replicates run in duplicates. **p<0.005; ***p<0.001.

Techniques: Activity Assay, Expressing, Western Blot, Cytotoxicity Assay

EphA2 interacts with liprinα–1 and vinculin. (A) EphA2 interactome involved in cell motility as identified by proteomics. (B) Co-immunoprecipitation and western immunoblot of serum-starved T24 cells ±progranulin (150 nM) with the indicated antibodies. (C) Immunoblot of Liprinα–1 and vinculin in various urothelial carcinoma cells. (D) Co-localization of EphA2/liprinα–1 assessed by confocal microscopy. Insets = higher magnification of the relative areas within the white boxes. Bar = 10 μm. (E) Western immunoblot of liprinα–1- depleted T24 cells. (F) Quantification of T24 cell lateral motility determined by wound healing assays as previously described [18,19,26]. p = 0.000666.

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Progranulin/EphA2 axis: A novel oncogenic mechanism in bladder cancer

doi: 10.1016/j.matbio.2020.03.009

Figure Lengend Snippet: EphA2 interacts with liprinα–1 and vinculin. (A) EphA2 interactome involved in cell motility as identified by proteomics. (B) Co-immunoprecipitation and western immunoblot of serum-starved T24 cells ±progranulin (150 nM) with the indicated antibodies. (C) Immunoblot of Liprinα–1 and vinculin in various urothelial carcinoma cells. (D) Co-localization of EphA2/liprinα–1 assessed by confocal microscopy. Insets = higher magnification of the relative areas within the white boxes. Bar = 10 μm. (E) Western immunoblot of liprinα–1- depleted T24 cells. (F) Quantification of T24 cell lateral motility determined by wound healing assays as previously described [18,19,26]. p = 0.000666.

Techniques: Immunoprecipitation, Western Blot, Confocal Microscopy

Mechanism of progranulin-dependent EphA2 activation. Scheme depicting progranulin-induced EphA2 activation at Tyr588 and resultant Akt/MAPK feedback loop to phosphorylate Ser897.

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Progranulin/EphA2 axis: A novel oncogenic mechanism in bladder cancer

doi: 10.1016/j.matbio.2020.03.009

Figure Lengend Snippet: Mechanism of progranulin-dependent EphA2 activation. Scheme depicting progranulin-induced EphA2 activation at Tyr588 and resultant Akt/MAPK feedback loop to phosphorylate Ser897.

Techniques: Activation Assay

EPHA2 receptor consists of the ligand-binding domain, a cysteine-rich domain, two fibronectin-III domains, a tyrosine kinase (TK) domain, a sterile alpha motif (SAM), and a PDZ-binding motif. Novel mutations (yellow) and known SNPs were found in ligand-binding domain and TK domain by us recently

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: EPHA2 receptor consists of the ligand-binding domain, a cysteine-rich domain, two fibronectin-III domains, a tyrosine kinase (TK) domain, a sterile alpha motif (SAM), and a PDZ-binding motif. Novel mutations (yellow) and known SNPs were found in ligand-binding domain and TK domain by us recently

Article Snippet: EPHA2 expression levels were determined in whole-cell lysates by immunoblotting using anti-EPHA2 antibody (Santa Cruz Biotechnologies, Santa Cruz, CA).

Techniques: Ligand Binding Assay, Sterility, Binding Assay

Thirty five SCC, 39 MPM tumor samples, and six cell lines have been used to determine EPHA2 gene amplification. Fold change relative to reference gene LINE-1

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: Thirty five SCC, 39 MPM tumor samples, and six cell lines have been used to determine EPHA2 gene amplification. Fold change relative to reference gene LINE-1

Article Snippet: EPHA2 expression levels were determined in whole-cell lysates by immunoblotting using anti-EPHA2 antibody (Santa Cruz Biotechnologies, Santa Cruz, CA).

Techniques: Amplification

a Lysates of six MPM cell lines and the Met-5A, a mesothelial control cell line, were immunoblotted with EPHA2 antibody. b Immunohistochemistry representative pictures of 65 MPM tumor and nine normal mesothelium samples were used in MPM TMA. c Protein expression quantity of 65 MPM tumor and nine normal mesothelium samples were used in MPM TMA. d Protein expression quantity of 48 NSCLC SQ and 24 adjacent normal samples were used in SSC TMA. H&E, EPHA2, phospho-(p-)EPHA2, and ephrin A1 were stained and scored. N: normal, T: Tumor

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: a Lysates of six MPM cell lines and the Met-5A, a mesothelial control cell line, were immunoblotted with EPHA2 antibody. b Immunohistochemistry representative pictures of 65 MPM tumor and nine normal mesothelium samples were used in MPM TMA. c Protein expression quantity of 65 MPM tumor and nine normal mesothelium samples were used in MPM TMA. d Protein expression quantity of 48 NSCLC SQ and 24 adjacent normal samples were used in SSC TMA. H&E, EPHA2, phospho-(p-)EPHA2, and ephrin A1 were stained and scored. N: normal, T: Tumor

Article Snippet: EPHA2 expression levels were determined in whole-cell lysates by immunoblotting using anti-EPHA2 antibody (Santa Cruz Biotechnologies, Santa Cruz, CA).

Techniques: Control, Immunohistochemistry, Expressing, Staining

BEAS2B EPHA2 isogenic cells were used to treat a Taxol, b cisplatin, c SU11274, and d rapamycin. Mutation G391R cells showed resistant to cisplatin inhibition but sensitive to MET inhibitor SU11274 and mTOR inhibitor Rapamycin. EV: empty vector

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: BEAS2B EPHA2 isogenic cells were used to treat a Taxol, b cisplatin, c SU11274, and d rapamycin. Mutation G391R cells showed resistant to cisplatin inhibition but sensitive to MET inhibitor SU11274 and mTOR inhibitor Rapamycin. EV: empty vector

Article Snippet: EPHA2 expression levels were determined in whole-cell lysates by immunoblotting using anti-EPHA2 antibody (Santa Cruz Biotechnologies, Santa Cruz, CA).

Techniques: Mutagenesis, Inhibition, Plasmid Preparation

a MPM cell lines H28, H513, H2052, H2373, H2461, and H2596 were treated with cisplatin with 1, 5, and 10 μM for 48 h. b HEK293 EPHA2 isogenic cells treatment with doxazosin for 48 h

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: a MPM cell lines H28, H513, H2052, H2373, H2461, and H2596 were treated with cisplatin with 1, 5, and 10 μM for 48 h. b HEK293 EPHA2 isogenic cells treatment with doxazosin for 48 h

Article Snippet: EPHA2 expression levels were determined in whole-cell lysates by immunoblotting using anti-EPHA2 antibody (Santa Cruz Biotechnologies, Santa Cruz, CA).

Techniques:

a The crystal structure of EphA2 in the auto-inhibited conformation. The distance between Y772 and K702 shown by dotted line is 20.3 Å, b The EphA2 conformation after MD simulations showing the minimum distance between Y772 and K702 which is 3.4 Å, c The distribution of the distance between Y772 and K702 in the wild type and mutants A859D and T647M, d The surface showing the ATP binding site in EphA2, and e The distribution of the volume in (Å 3 ) of the ATP-binding site for the wild type and the two mutants calculated from the snapshots of the MD simulations

Journal: Oncogenesis

Article Title: EPHA2 mutations with oncogenic characteristics in squamous cell lung cancer and malignant pleural mesothelioma

doi: 10.1038/s41389-019-0159-6

Figure Lengend Snippet: a The crystal structure of EphA2 in the auto-inhibited conformation. The distance between Y772 and K702 shown by dotted line is 20.3 Å, b The EphA2 conformation after MD simulations showing the minimum distance between Y772 and K702 which is 3.4 Å, c The distribution of the distance between Y772 and K702 in the wild type and mutants A859D and T647M, d The surface showing the ATP binding site in EphA2, and e The distribution of the volume in (Å 3 ) of the ATP-binding site for the wild type and the two mutants calculated from the snapshots of the MD simulations

Article Snippet: EPHA2 expression levels were determined in whole-cell lysates by immunoblotting using anti-EPHA2 antibody (Santa Cruz Biotechnologies, Santa Cruz, CA).

Techniques: Binding Assay

Review

Structured Review

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1) Product Images from "Complexity of progranulin mechanisms of action in mesothelioma."

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