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R&D Systems anti epha2 n terminal mab
Anti Epha2 N Terminal Mab, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 94 stars, based on 30 article reviews

anti epha2 n terminal mab - by Bioz Stars, 2026-10

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Article Title: Proteolysis of EphA2 converts it from a tumor suppressor to an oncoprotein
Article Snippet: Anti-FLAG mouse mAb was purchased from Sigma (St. Louis, MO), and anti-c-myc mAb was from Roche Applied Science (Penzberg, Germany); anti-EphA2 (N-terminal) mAb was from R&D Systems (Minneapolis, MN); anti-EphA2 (C-terminal) pAb) and anti-phospho-Erk1/2 (p-T 202 /p-Y 204 ) mAb, anti-Erk pAb, anti-EphA2 mAb were from Santa Cruz Biotechnology (Dallas, TX); anti-Akt mAb and phospho-Akt (p-S 473 ) mAb were from Cell Signaling Technology (Danvers, MA); anti-phospho-EphA2 (p-S 897 ) pAb and anti-phospho-EphA2 (p-Y 594 ) pAb were from Cell Applications (San Diego, CA); anti-phospho-Y mAb (4G10) and anti-actin mAb were from Millipore (Billerica, MA).

Article Title: Proteolysis of EphA2 converts it from a tumor suppressor to an oncoprotein
Article Snippet: Antibodies and reagents Anti-FLAG mouse mAb was purchased from Sigma (St. Louis, MO), and anti-c-myc mAb was from Roche Applied Science (Penzberg, Germany); anti-EphA2 (N-terminal) mAb was from R&D Systems (Minneapolis, MN); anti-EphA2 (C-terminal) pAb) and anti-phospho-Erk1/2 (p-T 202 /p-Y 204 ) mAb, anti-Erk pAb, anti-EphA2 mAb were from Santa Cruz Biotechnology (Dallas, TX); anti-Akt mAb and phospho-Akt (p-S 473 ) mAb were from Cell Signaling Technology (Danvers, MA); anti-phospho-EphA2 (p-S 897 ) pAb and anti-phospho-EphA2 (p-Y 594 ) pAb were from Cell Applications (San Diego, CA); anti-phospho-Y mAb (4G10) and anti-actin mAb were from Millipore (Billerica, MA).

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(A, B) HeLa cells were pretreated with control or rhEphrin-A1 (5 μg/ml) for 1 h at 37°C and infected with Ctr (MOI-100) for 1 h at 4°C. Cells were fixed and immunostained for Ctr (Hsp60, green) and EphA2 (EphA2, red). (A) Images show EphA2 or EB or the co-localization of EB with EphA2 (yellow) on the cell surface. (B) Co-localization of EB with EphA2 was quantified from 8 different fields of view in 3 independent experiments. The data are expressed as a mean percentage of EphA2-associated EB (± SD) compared to total EB. *P<0.05. Error bars show mean ± SD. (C, D) Purified EB was incubated with or without rhEphA2 (5 μg/ml) in a serum free RPMI media containing 0.1% BSA for 1 h at 37°C. Additionally, rhEphrin-A1-preincubated rhEphA2 was incubated with EB as above. The mixture was centrifuged onto coverslips, washed 3 times with ice cold-PBS, stained and quantified as (B). The data are expressed as a mean percentage of EphA2-associated EB (± SD) compared to total EB. *P<0.05, **P<0.01. Error bars show mean ± SD. (E) Pull-down assay: Purified EB was incubated with rhEphA2 or with rhEphrin-A1 each 10 μg/ml in a serum free RPMI media containing 0.1% BSA for 45 min at 37°C in a rotary shaker. Additionally, rhEphrin-A1-preincubated rhEphA2 was incubated with EB as above and the mixture was centrifuged at 5000 x g for 4 min at 4°C. The pellet was gently washed 3 times with ice cold-PBS. Ctr -infected cells act as a positive control for full length EphA2 expression. The samples were subjected to WB analysis to determine the full length endogenous EphA2 (~130 kDa) and rhEphA2 (~70 kDa) using N-terminal EphA2 specific antibody. rhEphrin-A1 was expressed at ~55–60 kDa. Hsp60 acts as a control for EB. (F) EB pre-incubated with rhPHB (PHB-His) (PHB is a mitochondrial inner membrane protein. rhPHB acts as a control for His-tagged rhEphA2) or with rhEphA2 (EphA2-His) each 5 μg/ml for 1 h at 4°C were added to HeLa cells for 1 h at 4°C. Cells were fixed and immunostained for extracellular EB and Actin filaments (Phalloidin). The number of extracellular EB was counted randomly from 40 different cells. Data are expressed as percentage of extracellular EB relative to control. Shown is the mean ± SD of three independent experiments normalized to control+EB. *P<0.05. Error bars show mean ± SD. (G) HeLa cells were incubated with antibody against N-terminal-EphA2 or-PDI or-EphB4 each 10 μg/ml for 1 h at 4°C. Cells were then infected with GFP-expressing Ctr for 1 h at 4°C. Cells were fixed and immunostained against Actin filaments. Graph was made similar to (F). Shown is the mean ± SD of two independent experiments normalized to IgG control. *P<0.05, **P<0.01, ***P<0.001. Error bars show mean ± SD. (A, C) Magnification is indicated in size bar.

Journal: PLoS Pathogens

Article Title: EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

doi: 10.1371/journal.ppat.1004846

Figure Lengend Snippet: (A, B) HeLa cells were pretreated with control or rhEphrin-A1 (5 μg/ml) for 1 h at 37°C and infected with Ctr (MOI-100) for 1 h at 4°C. Cells were fixed and immunostained for Ctr (Hsp60, green) and EphA2 (EphA2, red). (A) Images show EphA2 or EB or the co-localization of EB with EphA2 (yellow) on the cell surface. (B) Co-localization of EB with EphA2 was quantified from 8 different fields of view in 3 independent experiments. The data are expressed as a mean percentage of EphA2-associated EB (± SD) compared to total EB. *P<0.05. Error bars show mean ± SD. (C, D) Purified EB was incubated with or without rhEphA2 (5 μg/ml) in a serum free RPMI media containing 0.1% BSA for 1 h at 37°C. Additionally, rhEphrin-A1-preincubated rhEphA2 was incubated with EB as above. The mixture was centrifuged onto coverslips, washed 3 times with ice cold-PBS, stained and quantified as (B). The data are expressed as a mean percentage of EphA2-associated EB (± SD) compared to total EB. *P<0.05, **P<0.01. Error bars show mean ± SD. (E) Pull-down assay: Purified EB was incubated with rhEphA2 or with rhEphrin-A1 each 10 μg/ml in a serum free RPMI media containing 0.1% BSA for 45 min at 37°C in a rotary shaker. Additionally, rhEphrin-A1-preincubated rhEphA2 was incubated with EB as above and the mixture was centrifuged at 5000 x g for 4 min at 4°C. The pellet was gently washed 3 times with ice cold-PBS. Ctr -infected cells act as a positive control for full length EphA2 expression. The samples were subjected to WB analysis to determine the full length endogenous EphA2 (~130 kDa) and rhEphA2 (~70 kDa) using N-terminal EphA2 specific antibody. rhEphrin-A1 was expressed at ~55–60 kDa. Hsp60 acts as a control for EB. (F) EB pre-incubated with rhPHB (PHB-His) (PHB is a mitochondrial inner membrane protein. rhPHB acts as a control for His-tagged rhEphA2) or with rhEphA2 (EphA2-His) each 5 μg/ml for 1 h at 4°C were added to HeLa cells for 1 h at 4°C. Cells were fixed and immunostained for extracellular EB and Actin filaments (Phalloidin). The number of extracellular EB was counted randomly from 40 different cells. Data are expressed as percentage of extracellular EB relative to control. Shown is the mean ± SD of three independent experiments normalized to control+EB. *P<0.05. Error bars show mean ± SD. (G) HeLa cells were incubated with antibody against N-terminal-EphA2 or-PDI or-EphB4 each 10 μg/ml for 1 h at 4°C. Cells were then infected with GFP-expressing Ctr for 1 h at 4°C. Cells were fixed and immunostained against Actin filaments. Graph was made similar to (F). Shown is the mean ± SD of two independent experiments normalized to IgG control. *P<0.05, **P<0.01, ***P<0.001. Error bars show mean ± SD. (A, C) Magnification is indicated in size bar.

Article Snippet: Antibodies against N-terminal EphA2 (D4A2) for WB and IF studies, pEphA2 (phospho EphA2 Ser897), pERK, pAkt, pPI3K, p85-PI3K, total Akt and total ERK were from Cell Signaling technology.

Techniques: Control, Infection, Purification, Incubation, Staining, Pull Down Assay, Positive Control, Expressing, Membrane

(A) Adherence assay: HeLa cells were transfected with siRNA against luciferase gene (siLuci) or EphA2 gene (siEphA2) for 40 h at 37°C. The transfected cells were infected with Ctr (MOI-100) for 1 h at 4°C followed by immunostaining Ctr -EB (Hsp60, green) and pEphA2 (phospho EphA2 Ser897, red). (B) Co-localization of EB with pEphA2 was quantified. The graph was made similar to . The data are expressed as a mean percentage of pEphA2-associated EB (± SD) compared to total EB. *P<0.05. Error bars show mean ± SD. (C) Invasion assay: The transfected cells were infected with Ctr (MOI-20-25) for 4–5 h at 35°C followed by immunostaining as (A) including Actin filaments. (A, C) Arrows indicate the co-localization of Ctr with pEphA2 (yellow). Magnification is indicated in size bar.

Journal: PLoS Pathogens

Article Title: EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

doi: 10.1371/journal.ppat.1004846

Figure Lengend Snippet: (A) Adherence assay: HeLa cells were transfected with siRNA against luciferase gene (siLuci) or EphA2 gene (siEphA2) for 40 h at 37°C. The transfected cells were infected with Ctr (MOI-100) for 1 h at 4°C followed by immunostaining Ctr -EB (Hsp60, green) and pEphA2 (phospho EphA2 Ser897, red). (B) Co-localization of EB with pEphA2 was quantified. The graph was made similar to . The data are expressed as a mean percentage of pEphA2-associated EB (± SD) compared to total EB. *P<0.05. Error bars show mean ± SD. (C) Invasion assay: The transfected cells were infected with Ctr (MOI-20-25) for 4–5 h at 35°C followed by immunostaining as (A) including Actin filaments. (A, C) Arrows indicate the co-localization of Ctr with pEphA2 (yellow). Magnification is indicated in size bar.

Techniques: Transfection, Luciferase, Infection, Immunostaining, Invasion Assay

(A) HeLa cells uninfected (UN) or infected with Ctr (MOI-50-75) for 30 min or 3 h were analyzed via FACS for surface EphA2 under non permeabilised or total EphA2 under permeabilised condition. Controls were indicated on the left curve chart and corresponding samples on the right curve chart of the same experiment. (UN: uninfected, Iso: isotype and perm: permeabilised). (B) Result of experiment shown in A demonstrated as bar chart. Shown is the mean ± SD of three independent experiments normalized to UN perm. *P<0.05, ns: non-significant. Error bars show mean ± SD. (C) HeLa cells were UN or infected with Ctr (MOI-50) for the indicated time points. The cells were immunobloted against pEphA2 and Actin. The blot was stripped and reprobed for total EphA2. (D) HeLa cells were treated with control or rhEphrin-A1 for 3 h and were harvested for WB analysis. (E) HeLa cells were UN or infected with EB for the indicated time points and immunoprecipitated (IP) with α-EphA2 or α-p85-PI3K antibodies. The IP material was solved in 40 μl Laemmli (100%) and loaded 20 μl for WB studies (50%) or (F) immunostained using α-EphA2 and α-pPI3K for 2 h at RT followed by secondary staining with anti-mouse Alexa fluor 488 and anti-rabbit Alexa fluor 647 for the microscopic analysis, respectively. Magnification is indicated in size bar.

Journal: PLoS Pathogens

Article Title: EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

doi: 10.1371/journal.ppat.1004846

Figure Lengend Snippet: (A) HeLa cells uninfected (UN) or infected with Ctr (MOI-50-75) for 30 min or 3 h were analyzed via FACS for surface EphA2 under non permeabilised or total EphA2 under permeabilised condition. Controls were indicated on the left curve chart and corresponding samples on the right curve chart of the same experiment. (UN: uninfected, Iso: isotype and perm: permeabilised). (B) Result of experiment shown in A demonstrated as bar chart. Shown is the mean ± SD of three independent experiments normalized to UN perm. *P<0.05, ns: non-significant. Error bars show mean ± SD. (C) HeLa cells were UN or infected with Ctr (MOI-50) for the indicated time points. The cells were immunobloted against pEphA2 and Actin. The blot was stripped and reprobed for total EphA2. (D) HeLa cells were treated with control or rhEphrin-A1 for 3 h and were harvested for WB analysis. (E) HeLa cells were UN or infected with EB for the indicated time points and immunoprecipitated (IP) with α-EphA2 or α-p85-PI3K antibodies. The IP material was solved in 40 μl Laemmli (100%) and loaded 20 μl for WB studies (50%) or (F) immunostained using α-EphA2 and α-pPI3K for 2 h at RT followed by secondary staining with anti-mouse Alexa fluor 488 and anti-rabbit Alexa fluor 647 for the microscopic analysis, respectively. Magnification is indicated in size bar.

Techniques: Infection, Control, Immunoprecipitation, Staining

(A) HeLa cells were infected with Ctr- EB (MOI-1) for 14, 20 and 26 h and cells were analyzed via FACS as . (B) HeLa cells were infected with Ctr- EB for 14, 24 and 36 h. Input (5%) was taken after homogenization step (before plasma membrane protein isolation) and subjected to WB analysis to determine pEphA2, Hsp60 and Actin. The blot was stripped and reprobed for total EphA2. Numbers under the blot represents fold activation for pEphA2 with respect to total EphA2. (C) Plasma membrane protein was isolated from UN or Ctr -infected cells at different time points and subjected to WB analysis. Pan Cadherin was used as a control for plasma membrane. IncA was used as a marker to test the quality of the isolated plasma membrane. * Unspecific bands detected by IncA polyclonal serum. (D) Culture medium of the UN as well as time course Ctr- infected cells (as indicated) was collected and TCA precipitated. The precipitated lysates were subjected to WB analysis against pEphA2, total EphA2 and GP96. (E, F) HeLa cells were transfected with EphA2-pcDNA3 at 37°C and the cells were infected with Ctr -pIncA-flag for 24 h at 35°C. (E) Cells were fixed and stained against Flag (red), EphA2 (green) and DNA (blue). (F) Cells were fixed and stained against Flag (red), pEphA2 (green) and DNA (blue). Magnification is indicated in size bar.

Journal: PLoS Pathogens

Article Title: EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

doi: 10.1371/journal.ppat.1004846

Figure Lengend Snippet: (A) HeLa cells were infected with Ctr- EB (MOI-1) for 14, 20 and 26 h and cells were analyzed via FACS as . (B) HeLa cells were infected with Ctr- EB for 14, 24 and 36 h. Input (5%) was taken after homogenization step (before plasma membrane protein isolation) and subjected to WB analysis to determine pEphA2, Hsp60 and Actin. The blot was stripped and reprobed for total EphA2. Numbers under the blot represents fold activation for pEphA2 with respect to total EphA2. (C) Plasma membrane protein was isolated from UN or Ctr -infected cells at different time points and subjected to WB analysis. Pan Cadherin was used as a control for plasma membrane. IncA was used as a marker to test the quality of the isolated plasma membrane. * Unspecific bands detected by IncA polyclonal serum. (D) Culture medium of the UN as well as time course Ctr- infected cells (as indicated) was collected and TCA precipitated. The precipitated lysates were subjected to WB analysis against pEphA2, total EphA2 and GP96. (E, F) HeLa cells were transfected with EphA2-pcDNA3 at 37°C and the cells were infected with Ctr -pIncA-flag for 24 h at 35°C. (E) Cells were fixed and stained against Flag (red), EphA2 (green) and DNA (blue). (F) Cells were fixed and stained against Flag (red), pEphA2 (green) and DNA (blue). Magnification is indicated in size bar.

Techniques: Infection, Homogenization, Clinical Proteomics, Membrane, Isolation, Activation Assay, Control, Marker, Transfection, Staining

(A) Infectivity assay performed for B) C) D) E) F): The transfected cells infected with Ctr (MOI-1) for 24 h at 35°C was referred as primary infection. The supernatant of the primary infected cells was taken to infect the fresh cells to determine the secondary infection (see -methods). (B) HeLa cells were left untransfected (control) or transfected with empty pcDNA3 or EphA2-pcDNA3 each 1 μg/ml and then left UN or infected with Ctr for 24 h. Cells were harvested for WB analysis. (C) HeLa cells were untransfected (control) or transfected with siRNA against luciferase gene (siLuci) or EphA2 gene (siEphA2) for 40 h at 37°C and then left UN or infected with Ctr for another 24 h. Cells were harvested for WB analysis. (D, E) Primary infected cell lysates of (B) and (C) were taken to infect the fresh cells for secondary infection (Infectivity assay). (F) Number of the inclusion per cell (%) for the infectivity assay was determined by counting the inclusion on 10 independent fields. Shown is the mean ± SD of three independent experiments normalized to control. **P<0.01. Error bars show mean ± SD. (G) Cells were transfected with siRNA against luciferase (siLuci) or EphA2 (siEphA2) or PDGFRβ (siPDGFRβ) or both together for 40 h at 37°C and then infected with Ctr . UN: uninfected cells. Cells were harvested to determine the respective proteins by WB analysis. (H) Size of the inclusion per cell (%) for (F) was determined by ImageJ software by measuring cells out of four microscopic fields. Shown is the mean ± SD of two independent experiments normalized to siLuci. *P<0.05. Error bars show mean ± SD. In all of the above experiments, numbers under the blot represents fold activation for phospho specific proteins with respect to total proteins and fold change for the total proteins.

Journal: PLoS Pathogens

Article Title: EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

doi: 10.1371/journal.ppat.1004846

Figure Lengend Snippet: (A) Infectivity assay performed for B) C) D) E) F): The transfected cells infected with Ctr (MOI-1) for 24 h at 35°C was referred as primary infection. The supernatant of the primary infected cells was taken to infect the fresh cells to determine the secondary infection (see -methods). (B) HeLa cells were left untransfected (control) or transfected with empty pcDNA3 or EphA2-pcDNA3 each 1 μg/ml and then left UN or infected with Ctr for 24 h. Cells were harvested for WB analysis. (C) HeLa cells were untransfected (control) or transfected with siRNA against luciferase gene (siLuci) or EphA2 gene (siEphA2) for 40 h at 37°C and then left UN or infected with Ctr for another 24 h. Cells were harvested for WB analysis. (D, E) Primary infected cell lysates of (B) and (C) were taken to infect the fresh cells for secondary infection (Infectivity assay). (F) Number of the inclusion per cell (%) for the infectivity assay was determined by counting the inclusion on 10 independent fields. Shown is the mean ± SD of three independent experiments normalized to control. **P<0.01. Error bars show mean ± SD. (G) Cells were transfected with siRNA against luciferase (siLuci) or EphA2 (siEphA2) or PDGFRβ (siPDGFRβ) or both together for 40 h at 37°C and then infected with Ctr . UN: uninfected cells. Cells were harvested to determine the respective proteins by WB analysis. (H) Size of the inclusion per cell (%) for (F) was determined by ImageJ software by measuring cells out of four microscopic fields. Shown is the mean ± SD of two independent experiments normalized to siLuci. *P<0.05. Error bars show mean ± SD. In all of the above experiments, numbers under the blot represents fold activation for phospho specific proteins with respect to total proteins and fold change for the total proteins.

Techniques: Infection, Transfection, Control, Luciferase, Software, Activation Assay

(A) HeLa cells were left UN or infected with Ctr (MOI-2) for the indicated period of times and immunoprecipitated using α-EphA2 antibody. The IP material was solved in 40 μl Laemmli (100%) and loaded 20 μl for WB studies (50%). (B) Cells were transfected with EphA2-pcDNA3 and p85-PI3K expression plasmid together for 15 h followed by Ctr infection for 24 h. Cells were stained against EphA2 (red), DNA (blue) and pPI3K (green). (C) Schematic representation of full length EphA2 plasmid, EphA2 kinase dead mutant plasmid (EphA2K645R) and EphA2 mutant plasmid missing the entire cytoplasmic domain (EphA2ΔIC). TM: transmembrane domain. (D) The plasmids having full length EphA2 or EphA2ΔIC were transfected and these cells were infected with Ctr for 20 h. Cells were fixed and stained against α-EphA2 antibody (red) and Ctr using α-Hsp60 (green). (E) Arrows were marked to illustrate the difference between the size of the inclusion of untransfected (white arrows) and transfected (red arrows) cells. (F) Size of the inclusion for (E) was determined by ImageJ software. Empty-plasmid transfected cells act as a control. Shown is the mean ± SD of two independent experiments normalized to empty-plasmid transfected cells. *P<0.05, **P<0.01. Error bars show mean ± SD. (G) Cells after transfection of the constructs (each 1.5 μg/ml) indicated above the lanes followed by infection (MOI-1) for 18 h were subjected to WB analysis to analyze the proteins as indicated. EphA2ΔIC was detected by N-terminal EphA2 antibody. Due to the high intensity of total EphA2 and phospho EphA2 from the EphA2-transfected infected cells, the upregulation of endogenous EphA2 cannot be visualized. (B, D, F) Magnification is indicated in size bar.

Journal: PLoS Pathogens

Article Title: EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

doi: 10.1371/journal.ppat.1004846

Figure Lengend Snippet: (A) HeLa cells were left UN or infected with Ctr (MOI-2) for the indicated period of times and immunoprecipitated using α-EphA2 antibody. The IP material was solved in 40 μl Laemmli (100%) and loaded 20 μl for WB studies (50%). (B) Cells were transfected with EphA2-pcDNA3 and p85-PI3K expression plasmid together for 15 h followed by Ctr infection for 24 h. Cells were stained against EphA2 (red), DNA (blue) and pPI3K (green). (C) Schematic representation of full length EphA2 plasmid, EphA2 kinase dead mutant plasmid (EphA2K645R) and EphA2 mutant plasmid missing the entire cytoplasmic domain (EphA2ΔIC). TM: transmembrane domain. (D) The plasmids having full length EphA2 or EphA2ΔIC were transfected and these cells were infected with Ctr for 20 h. Cells were fixed and stained against α-EphA2 antibody (red) and Ctr using α-Hsp60 (green). (E) Arrows were marked to illustrate the difference between the size of the inclusion of untransfected (white arrows) and transfected (red arrows) cells. (F) Size of the inclusion for (E) was determined by ImageJ software. Empty-plasmid transfected cells act as a control. Shown is the mean ± SD of two independent experiments normalized to empty-plasmid transfected cells. *P<0.05, **P<0.01. Error bars show mean ± SD. (G) Cells after transfection of the constructs (each 1.5 μg/ml) indicated above the lanes followed by infection (MOI-1) for 18 h were subjected to WB analysis to analyze the proteins as indicated. EphA2ΔIC was detected by N-terminal EphA2 antibody. Due to the high intensity of total EphA2 and phospho EphA2 from the EphA2-transfected infected cells, the upregulation of endogenous EphA2 cannot be visualized. (B, D, F) Magnification is indicated in size bar.

Techniques: Infection, Immunoprecipitation, Transfection, Expressing, Plasmid Preparation, Staining, Mutagenesis, Software, Control, Construct

(A) HUVEC cells were left uninfected or infected with Ctr (MOI-1) for 14 h followed by treatment with DMSO or PI3K inhibitor LY294002 (15 μM) or MAPK inhibitor UO126 (15 μM) for 10 h. After 24 h of total infection, cells were subjected to WB analysis against respective proteins. (B) EphA2 plasmid-transfected HeLa cells were left uninfected or infected with Ctr for 24 h as mentioned above the lanes. The cells were treated with DMSO or UO126 (30 μM) for 3 h or 6 h as described above the lanes and subjected to WB analysis to determine the respective proteins as indicated.

Journal: PLoS Pathogens

Article Title: EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

doi: 10.1371/journal.ppat.1004846

Figure Lengend Snippet: (A) HUVEC cells were left uninfected or infected with Ctr (MOI-1) for 14 h followed by treatment with DMSO or PI3K inhibitor LY294002 (15 μM) or MAPK inhibitor UO126 (15 μM) for 10 h. After 24 h of total infection, cells were subjected to WB analysis against respective proteins. (B) EphA2 plasmid-transfected HeLa cells were left uninfected or infected with Ctr for 24 h as mentioned above the lanes. The cells were treated with DMSO or UO126 (30 μM) for 3 h or 6 h as described above the lanes and subjected to WB analysis to determine the respective proteins as indicated.

Techniques: Infection, Plasmid Preparation, Transfection

(A) The transfection efficiency of siRNA directed against EphA2 was monitored by WB analysis against total EphA2. In addition, levels of pAkt, IncA and Actin were verified. (B) siLuci or siEphA2-transfected cells were left uninfected or infected with Ctr for 16 h. Cells were induced to apoptosis by TNF-α (50 ng/ml)/CHX (5 μg/ml) for 5–6 h. Processing of PARP, Hsp60 and Actin were monitored by WB analysis. Rectangle boxes (black: before infection) or (red: after infection) denotes the difference in PARP cleavage after TNF-α induction in siLuci and siEphA2 transfected cells. (C) For quantification, TUNEL positive cells from each sample were counted from ten different fields. Shown is the mean ± SD of two independent experiments. **P<0.01, *P<0.05, ns: non-significant. Error bars show mean ± SD.

Journal: PLoS Pathogens

Article Title: EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

doi: 10.1371/journal.ppat.1004846

Figure Lengend Snippet: (A) The transfection efficiency of siRNA directed against EphA2 was monitored by WB analysis against total EphA2. In addition, levels of pAkt, IncA and Actin were verified. (B) siLuci or siEphA2-transfected cells were left uninfected or infected with Ctr for 16 h. Cells were induced to apoptosis by TNF-α (50 ng/ml)/CHX (5 μg/ml) for 5–6 h. Processing of PARP, Hsp60 and Actin were monitored by WB analysis. Rectangle boxes (black: before infection) or (red: after infection) denotes the difference in PARP cleavage after TNF-α induction in siLuci and siEphA2 transfected cells. (C) For quantification, TUNEL positive cells from each sample were counted from ten different fields. Shown is the mean ± SD of two independent experiments. **P<0.01, *P<0.05, ns: non-significant. Error bars show mean ± SD.

Techniques: Transfection, Infection, TUNEL Assay

(A) HeLa cells left UN or infected with Ctr -serovar D were centrifuged at 910 x g for 30 min and allowed to infect for the indicated time points. The cells were harvested and subjected to WB analysis. (B) HeLa cells were transfected with siRNA against luciferase gene (siLuci) or EphA2 gene (siEphA2) for 40 h at 37°C. Adherence assay: The transfected cells were infected with Ctr- serovar D for 1 h at 4°C. Cells were immunostained against EB and Actin filaments. Number of extracellular EB was counted randomly from 30 different cells. Data are expressed as percentage of extracellular EB relative to siLuci. Shown is the mean ± SD of three independent experiments normalized to siLuci. *P<0.05. Error bars show mean ± SD. Invasion assay: The transfected cells were infected with Ctr- serovar D for 4–5 h at 35°C. Cells were immunostained against EB and Actin filaments. Number of invaded EB was counted randomly from 30 different cells. Data are expressed as percentage of invaded EB relative to siLuci. Shown is the mean ± SD of three independent experiments normalized to siLuci. *P<0.001. Error bars show mean ± SD. (C) FACS analysis was performed with Ctr -serovar D infected cells as . (UN: uninfected, Iso: isotype and perm: permeabilised). (D) Results of C were shown as bar charts. Shown is the mean ± SD of two independent experiments normalized to UN. ***P<0.001, *P<0.05, ns: non-significant. Error bars show mean ± SD. (E) HeLa cells were left UN or infected with Ctr- serovar D (MOI-1) for 14, 20 and 28 h and cells were analyzed via FACS as . (F) EphA2 knockdown followed by 16 h Ctr -serovar D infected cells were induced to apoptosis by TNF-α (50 ng/ml)/CHX (5 μg/ml) for 5–6 h. Processing of PARP, Hsp60 and Actin were monitored by WB analysis.

Journal: PLoS Pathogens

Article Title: EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

doi: 10.1371/journal.ppat.1004846

Figure Lengend Snippet: (A) HeLa cells left UN or infected with Ctr -serovar D were centrifuged at 910 x g for 30 min and allowed to infect for the indicated time points. The cells were harvested and subjected to WB analysis. (B) HeLa cells were transfected with siRNA against luciferase gene (siLuci) or EphA2 gene (siEphA2) for 40 h at 37°C. Adherence assay: The transfected cells were infected with Ctr- serovar D for 1 h at 4°C. Cells were immunostained against EB and Actin filaments. Number of extracellular EB was counted randomly from 30 different cells. Data are expressed as percentage of extracellular EB relative to siLuci. Shown is the mean ± SD of three independent experiments normalized to siLuci. *P<0.05. Error bars show mean ± SD. Invasion assay: The transfected cells were infected with Ctr- serovar D for 4–5 h at 35°C. Cells were immunostained against EB and Actin filaments. Number of invaded EB was counted randomly from 30 different cells. Data are expressed as percentage of invaded EB relative to siLuci. Shown is the mean ± SD of three independent experiments normalized to siLuci. *P<0.001. Error bars show mean ± SD. (C) FACS analysis was performed with Ctr -serovar D infected cells as . (UN: uninfected, Iso: isotype and perm: permeabilised). (D) Results of C were shown as bar charts. Shown is the mean ± SD of two independent experiments normalized to UN. ***P<0.001, *P<0.05, ns: non-significant. Error bars show mean ± SD. (E) HeLa cells were left UN or infected with Ctr- serovar D (MOI-1) for 14, 20 and 28 h and cells were analyzed via FACS as . (F) EphA2 knockdown followed by 16 h Ctr -serovar D infected cells were induced to apoptosis by TNF-α (50 ng/ml)/CHX (5 μg/ml) for 5–6 h. Processing of PARP, Hsp60 and Actin were monitored by WB analysis.

Techniques: Infection, Transfection, Luciferase, Invasion Assay, Knockdown

(1) Ctr -EB bind and activate EphA2 and enter the cell together with the activated receptor and PI3K recruited to EphA2. (2) Ctr -mediated ERK activation induces EphA2 upregulation. (3) Upregulated EphA2 does not appear at the cell surface. (4) Intracellular EphA2 is associated with the Ctr inclusion which interacts with pPI3K. (5, 6) EphA2-induced signaling cascades (pAkt) are required to inhibit apoptosis and enhance the infection.

Journal: PLoS Pathogens

Article Title: EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

doi: 10.1371/journal.ppat.1004846

Figure Lengend Snippet: (1) Ctr -EB bind and activate EphA2 and enter the cell together with the activated receptor and PI3K recruited to EphA2. (2) Ctr -mediated ERK activation induces EphA2 upregulation. (3) Upregulated EphA2 does not appear at the cell surface. (4) Intracellular EphA2 is associated with the Ctr inclusion which interacts with pPI3K. (5, 6) EphA2-induced signaling cascades (pAkt) are required to inhibit apoptosis and enhance the infection.

Techniques: Activation Assay, Infection

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