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Thermo Fisher e1b ap5
(A) Localization of <t>E1B-AP5,</t> ATRIP, and RPA32 in mock-infected interphase A549 cells. Cells were grown on glass coverslips and then treated with preextraction buffer and fixed with 4% (wt/vol) paraformaldehyde as described in Materials and Methods. Antigens were detected using the appropriate antibodies. (B) Localization of pTP, DBP, and RPA32 at viral replication centers in Ad5-infected A549 cells. Twenty-four hours postinfection with 10 PFU/cell wt Ad5, cells were treated with preextraction buffer and then fixed with 4% (wt/vol) paraformaldehyde, whereupon antigens were detected using the appropriate reagents. Colocalization images were recorded using a Zeiss LSM510-Meta laser scanning confocal microscope. Nuclei are stained with DAPI and are shown in blue.
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1) Product Images from "A Role for E1B-AP5 in ATR Signaling Pathways during Adenovirus Infection "

Article Title: A Role for E1B-AP5 in ATR Signaling Pathways during Adenovirus Infection

Journal: Journal of Virology

doi: 10.1128/JVI.00170-08

(A) Localization of E1B-AP5, ATRIP, and RPA32 in mock-infected interphase A549 cells. Cells were grown on glass coverslips and then treated with preextraction buffer and fixed with 4% (wt/vol) paraformaldehyde as described in Materials and Methods. Antigens were detected using the appropriate antibodies. (B) Localization of pTP, DBP, and RPA32 at viral replication centers in Ad5-infected A549 cells. Twenty-four hours postinfection with 10 PFU/cell wt Ad5, cells were treated with preextraction buffer and then fixed with 4% (wt/vol) paraformaldehyde, whereupon antigens were detected using the appropriate reagents. Colocalization images were recorded using a Zeiss LSM510-Meta laser scanning confocal microscope. Nuclei are stained with DAPI and are shown in blue.
Figure Legend Snippet: (A) Localization of E1B-AP5, ATRIP, and RPA32 in mock-infected interphase A549 cells. Cells were grown on glass coverslips and then treated with preextraction buffer and fixed with 4% (wt/vol) paraformaldehyde as described in Materials and Methods. Antigens were detected using the appropriate antibodies. (B) Localization of pTP, DBP, and RPA32 at viral replication centers in Ad5-infected A549 cells. Twenty-four hours postinfection with 10 PFU/cell wt Ad5, cells were treated with preextraction buffer and then fixed with 4% (wt/vol) paraformaldehyde, whereupon antigens were detected using the appropriate reagents. Colocalization images were recorded using a Zeiss LSM510-Meta laser scanning confocal microscope. Nuclei are stained with DAPI and are shown in blue.

Techniques Used: Infection, Microscopy, Staining

Effects of Ad infection on protein levels of E1B-55K binding proteins. A549 cells were infected with 10 PFU per cell of either wt Ad5 (A) or wt Ad12 (B). Cells were harvested at the appropriate times postinfection, and 50 μg protein samples was separated by SDS-PAGE and transferred onto nitrocellulose. Membranes were then Western blotted for E1B-AP5 (i), MRE11 (ii), p53 (iii), E1B-55K (iv), and β-actin (v) using the appropriate antibodies and visualized by enhanced chemiluminescence. (C) Immunofluorescent Zeiss LSM510-Meta confocal microscopic detection of E1B-AP5 protein expression in wt Ad5- and wt Ad12-infected A549 cells. Cells were infected with either 10 PFU/cell wt Ad5 or wt Ad12, subsequently fixed with 4% (wt/vol) paraformaldehyde in PBS, and permeabilized with acetone 24 h postinfection. E1B-AP5 (i and iv), Ad5 DBP (ii), and Ad12 E1B-55K (v) were then visualized using the appropriate antibodies. Nuclei are stained with DAPI and are shown in blue.
Figure Legend Snippet: Effects of Ad infection on protein levels of E1B-55K binding proteins. A549 cells were infected with 10 PFU per cell of either wt Ad5 (A) or wt Ad12 (B). Cells were harvested at the appropriate times postinfection, and 50 μg protein samples was separated by SDS-PAGE and transferred onto nitrocellulose. Membranes were then Western blotted for E1B-AP5 (i), MRE11 (ii), p53 (iii), E1B-55K (iv), and β-actin (v) using the appropriate antibodies and visualized by enhanced chemiluminescence. (C) Immunofluorescent Zeiss LSM510-Meta confocal microscopic detection of E1B-AP5 protein expression in wt Ad5- and wt Ad12-infected A549 cells. Cells were infected with either 10 PFU/cell wt Ad5 or wt Ad12, subsequently fixed with 4% (wt/vol) paraformaldehyde in PBS, and permeabilized with acetone 24 h postinfection. E1B-AP5 (i and iv), Ad5 DBP (ii), and Ad12 E1B-55K (v) were then visualized using the appropriate antibodies. Nuclei are stained with DAPI and are shown in blue.

Techniques Used: Infection, Binding Assay, SDS Page, Western Blot, Expressing, Staining

Colocalization of E1B-AP5 and RPA32, and ATRIP and RPA32, at Ad5 (A) and Ad12 (B) replication centers. Cells were infected with 10 PFU/cell of the appropriate virus. Ad5-infected and Ad12-infected cells were treated with preextraction buffer and then fixed with 4% (wt/vol) paraformaldehyde 24 h and 48 h postinfection, respectively. E1B-AP5, RPA32, and ATRIP localizations were visualized using the appropriate antibodies. Colocalization images were recorded using a Zeiss LSM510-Meta confocal microscope. Regions of substantial colocalization are shown as yellow in the merged image. Nuclei are stained with DAPI and are shown in blue.
Figure Legend Snippet: Colocalization of E1B-AP5 and RPA32, and ATRIP and RPA32, at Ad5 (A) and Ad12 (B) replication centers. Cells were infected with 10 PFU/cell of the appropriate virus. Ad5-infected and Ad12-infected cells were treated with preextraction buffer and then fixed with 4% (wt/vol) paraformaldehyde 24 h and 48 h postinfection, respectively. E1B-AP5, RPA32, and ATRIP localizations were visualized using the appropriate antibodies. Colocalization images were recorded using a Zeiss LSM510-Meta confocal microscope. Regions of substantial colocalization are shown as yellow in the merged image. Nuclei are stained with DAPI and are shown in blue.

Techniques Used: Infection, Microscopy, Staining

(A) Colocalization of E1B-AP5 with E1B-55K at Ad5 and Ad12 replication centers. Cells were infected with 10 PFU/cell of the appropriate virus. Infected cells were treated with preextraction buffer and then fixed with 4% (wt/vol) paraformaldehyde 24 h postinfection. E1B-AP5 and Ad E1B-55K were visualized using the appropriate antibodies. (B) Recruitment of E1B-AP5, RPA32, and ATRIP to Ad5 replication centers is independent of E1B-55K. Cells were infected with 10 PFU/cell of Ad5 dl 1520 and treated with preextraction buffer and then fixed with 4% (wt/vol) paraformaldehyde 24 h postinfection. E1B-AP5, Ad5 DBP, RPA32, and ATRIP were visualized using the appropriate antibodies. Colocalization images were recorded using a Zeiss LSM510-Meta confocal microscope. Regions of substantial colocalization are shown as yellow in the merged image. Nuclei are stained with DAPI and are shown in blue.
Figure Legend Snippet: (A) Colocalization of E1B-AP5 with E1B-55K at Ad5 and Ad12 replication centers. Cells were infected with 10 PFU/cell of the appropriate virus. Infected cells were treated with preextraction buffer and then fixed with 4% (wt/vol) paraformaldehyde 24 h postinfection. E1B-AP5 and Ad E1B-55K were visualized using the appropriate antibodies. (B) Recruitment of E1B-AP5, RPA32, and ATRIP to Ad5 replication centers is independent of E1B-55K. Cells were infected with 10 PFU/cell of Ad5 dl 1520 and treated with preextraction buffer and then fixed with 4% (wt/vol) paraformaldehyde 24 h postinfection. E1B-AP5, Ad5 DBP, RPA32, and ATRIP were visualized using the appropriate antibodies. Colocalization images were recorded using a Zeiss LSM510-Meta confocal microscope. Regions of substantial colocalization are shown as yellow in the merged image. Nuclei are stained with DAPI and are shown in blue.

Techniques Used: Infection, Microscopy, Staining

(A) Ad5 and Ad12 differentially regulate the phosphorylation of RPA32, Rad9, and Smc1 during infection. A549 cells were infected with 10 PFU/cell of either wt Ad5 or wt Ad12. Cells were harvested at the appropriate times postinfection, and 50 μg protein samples was separated by SDS-PAGE. After electrophoretic transfer onto nitrocellulose, membranes were Western blotted for RPA32 (i), RPA32 S4/8 (ii), Rad9 (iii), Smc1-S966 (iv), Smc1 (v), Chk1-S345 (vi), Chk1 (vii), γ-H2AX (viii), and H2AX (ix) using the appropriate antibodies. (B) E1B-AP5 is required for Ad12-induced phosphorylation of RPA32. A549 cells were initially treated with either nonsilencing (non-sil.) siRNA or siRNA oligonucleotides specific for the E1B-AP5 gene. Cells were subsequently infected with either Ad5 or Ad12 (at 10 PFU/cell), and whole-cell lysates were prepared at the appropriate times postinfection. After SDS-PAGE and transfer onto nitrocellulose, membranes were probed for E1B-AP5 (i), RPA32 (ii), RPA32 S4/8 (iii), Smc1-S966 (iv), Smc1 (v), γ-H2AX (vi), and H2AX (vii) with the appropriate antibodies. Antigens were visualized by ECL.
Figure Legend Snippet: (A) Ad5 and Ad12 differentially regulate the phosphorylation of RPA32, Rad9, and Smc1 during infection. A549 cells were infected with 10 PFU/cell of either wt Ad5 or wt Ad12. Cells were harvested at the appropriate times postinfection, and 50 μg protein samples was separated by SDS-PAGE. After electrophoretic transfer onto nitrocellulose, membranes were Western blotted for RPA32 (i), RPA32 S4/8 (ii), Rad9 (iii), Smc1-S966 (iv), Smc1 (v), Chk1-S345 (vi), Chk1 (vii), γ-H2AX (viii), and H2AX (ix) using the appropriate antibodies. (B) E1B-AP5 is required for Ad12-induced phosphorylation of RPA32. A549 cells were initially treated with either nonsilencing (non-sil.) siRNA or siRNA oligonucleotides specific for the E1B-AP5 gene. Cells were subsequently infected with either Ad5 or Ad12 (at 10 PFU/cell), and whole-cell lysates were prepared at the appropriate times postinfection. After SDS-PAGE and transfer onto nitrocellulose, membranes were probed for E1B-AP5 (i), RPA32 (ii), RPA32 S4/8 (iii), Smc1-S966 (iv), Smc1 (v), γ-H2AX (vi), and H2AX (vii) with the appropriate antibodies. Antigens were visualized by ECL.

Techniques Used: Infection, SDS Page, Western Blot

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Article Title: A Role for E1B-AP5 in ATR Signaling Pathways during Adenovirus Infection
Article Snippet: .. Small interfering RNA (siRNA) oligonucleotides targeting ATR and E1B-AP5 were purchased from Ambion, and their sequences were as follows: 5′-GGAAUAUAAUACAGUUGUATT-3′ (ATR) and 5′-GCAGUGGAACCAGUACUAUTT-3′ (E1B-AP5). .. As a negative control, AllStars negative control siRNA was purchased from Qiagen.