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1.
FIG. 4.

FIG. 4. From: The N Terminus of Adenovirus Type 12 E1A Inhibits Major Histocompatibility Complex Class I Expression by Preventing Phosphorylation of NF-?B p65 Ser276 through Direct Binding .

Sequence of the N terminus (residues 1 to 66) of E1A-12 that interacts with p65 and disables p65 from stimulating transcription. The shaded residues are completely unique to E1A-12, and the boxed residues with asterisks are chemically conserved among tumorigenic strains Ad40 and Ad41. The underlined residues comprise a portion of CR1.

Junfang Jiao, et al. J Virol. 2010 August;84(15):7668-7674.
2.
FIG. 7.

FIG. 7. From: The N Terminus of Adenovirus Type 12 E1A Inhibits Major Histocompatibility Complex Class I Expression by Preventing Phosphorylation of NF-?B p65 Ser276 through Direct Binding .

E1A-12 N-terminal region downregulates MHC-I. NIH 3T3 cells were transiently transfected with same amounts (15 μg) of empty vector (control) or plasmids expressing aa1-66, aa31-266, or wt E1A-12. At 72 h posttransfection, FACS analyses of MHC-I surface antigens were conducted. Data represent the means of fluorescence intensity of cell surface MHC-I staining from two independent FACS analyses.

Junfang Jiao, et al. J Virol. 2010 August;84(15):7668-7674.
3.
FIG. 6.

FIG. 6. From: The N Terminus of Adenovirus Type 12 E1A Inhibits Major Histocompatibility Complex Class I Expression by Preventing Phosphorylation of NF-?B p65 Ser276 through Direct Binding .

The N-terminal 40 amino acid residues of E1A-12 bind to p65 directly. The interaction between p65 and E1A-12 was determined by His6 pulldown assays. Recombinant His6-tagged WT E1A-12, aa1-40, or aa41-266 (1 μg each) was bound to Ni-NTA agarose beads, followed by incubation with 2 μg of recombinant GST-p65 or GST. After extensive washing, protein complexes were eluted with 300 mM imidazole and analyzed by Western blotting using p65 antibody (right panel) or GST antibody (left panel). As a negative control, the same amounts of GST-p65 or GST were incubated with Ni-NTA agarose beads that were not pretreated with His6-tagged E1A-12 proteins, followed by the same procedures as described above. Each input (lanes 1 and 6) contains approximately 20 ng of GST-p65 or GST.

Junfang Jiao, et al. J Virol. 2010 August;84(15):7668-7674.
4.
FIG. 1.

FIG. 1. From: The N Terminus of Adenovirus Type 12 E1A Inhibits Major Histocompatibility Complex Class I Expression by Preventing Phosphorylation of NF-?B p65 Ser276 through Direct Binding .

The N terminus of E1A-12 inhibits phosphorylation of p65-Ser276. (A) Schematic diagram showing wild-type E1A-12 and truncated E1A-12 mutants. The three conserved regions (CR1, CR2, and CR3) and a unique spacer region involved in tumorigenesis are indicated. (B) Constructs of N-terminally FLAG-tagged wt E1A-12, E1A-12 deletion mutants, or wt E1A-5 were cotransfected into COS-7 cells with a p65 plasmid. At 40 h posttransfection, whole-cell lysates were prepared and subjected to Western blot analysis using a phospho (P)-p65-Ser276 antibody (top panel). The same blot was stripped and reprobed with an antibody recognizing total (both phosphorylated and unphosphorylated) p65 (middle panel) and an antibody against the FLAG tag (lower panel). Nonspecific (NS) protein bands are boxed with dashed line.

Junfang Jiao, et al. J Virol. 2010 August;84(15):7668-7674.
5.
FIG. 2.

FIG. 2. From: The N Terminus of Adenovirus Type 12 E1A Inhibits Major Histocompatibility Complex Class I Expression by Preventing Phosphorylation of NF-?B p65 Ser276 through Direct Binding .

Transcriptional stimulation by p65 is suppressed by the N-terminal region of E1A-12. Plasmids expressing a Gal4-p65 fusion protein and luciferase reporters that contain a Gal4 binding site were cotransfected into COS-7 cells in the absence (bar 2) or the presence of FLAG-tagged E1A-12 wt (bar 3) and mutant (bars 4 to 8) proteins. Transfection with the plasmid expressing Gal4 alone served as a negative control (bar 1). Luciferase activity driven by p65 was measured at 40 h posttransfection. For each transfection assay, a Renilla luciferase reporter was included for normalization of gene expression. Each transfection was performed in duplicate and repeated two to three times. Error bars represent standard deviations. Cell lysates from transfections were subjected to Western blotting using anti-p65 and anti-FLAG antibodies to probe the levels of Gal4-p65 and E1A-12 proteins, respectively. Nonspecific (NS) protein bands are boxed with dashed line.

Junfang Jiao, et al. J Virol. 2010 August;84(15):7668-7674.
6.
FIG. 3.

FIG. 3. From: The N Terminus of Adenovirus Type 12 E1A Inhibits Major Histocompatibility Complex Class I Expression by Preventing Phosphorylation of NF-?B p65 Ser276 through Direct Binding .

The N-terminal region of E1A-12 is required to interact with p65 in vivo. (A) Coimmunoprecipitation of p65 by E1A-12 containing an intact N terminus. COS-7 cells were cotransfected with plasmids that express FLAG-tagged wt E1A-12, E1A-12 deletion mutants, or WT E1A-5. After 40 h, expressed E1A proteins were immunoprecipitated (IP) from cell lysates with a rabbit FLAG antibody, followed by Western blotting using a rabbit anti-p65 antibody. The interactions of p65 with wt E1A-12 (lane 3), aa1-240 (lane 6), aa1-66 (lane 9), aa31-266 (lane 12), aa95-266 (lane 15), and E1A-5 (lane 18) are shown. As a negative control, immunoprecipitation was performed with normal rabbit serum (lanes 2, 5, 8, 11, 14, and 17). p65 input (2% of whole-cell lysate) for each immunoprecipitation assay is also shown (lanes 1, 4, 7, 10, 13, and 16). The positions of p65 and IgG heavy chain are indicated. IgG ctrl, control IgG; IgG-h, IgG heavy chain. (B) Coimmunoprecipitation of E1A-12 that contains an intact N terminus by p65. Cell lysates in panel A were also subjected to reciprocal immunoprecipitation (goat α-p65) and Western blot (rabbit α-FLAG) analysis. The coprecipitated wt E1A-12 (lane 3), aa1-240 (lane 6), aa1-66 (lane 9), aa31-266 (lane 12), aa95-266 (lane 15), and E1A-5 (lane 18) are shown. Control immunoprecipitations were performed with normal goat serum (lanes 2, 5, 8, 11, 14, and 17). E1A input (2% of whole-cell lysate) for each immunoprecipitation assay is also shown (lanes 1, 4, 7, 10, 13, and 16).

Junfang Jiao, et al. J Virol. 2010 August;84(15):7668-7674.
7.
FIG. 5.

FIG. 5. From: The N Terminus of Adenovirus Type 12 E1A Inhibits Major Histocompatibility Complex Class I Expression by Preventing Phosphorylation of NF-?B p65 Ser276 through Direct Binding .

The N-terminal 40 amino acid residues of E1A-12 prevent PKAc from phosphorylating p65-Ser276 in vitro. (A) E1A-12 N-terminal 40-amino-acid peptide blocks p65-Ser276 phosphorylation in the PKAc assay. Recombinant His6-p65 (0.4 μg) was either left untreated or incubated with increasing amounts (0, 10, 20, and 50 μM) of the N-terminal 40-amino-acid peptide (pep1-40) of E1A12 in the presence of PKAc and ATP. Phosphorylation of p65 was examined by Western blotting with an antibody that specifically recognizes phosphorylated p65-Ser276. As a quantitative control, the same blot was probed with anti-p65 antibody that recognizes total p65. The pep1-40 peptide included in each assay was detected by sliver staining. (B) Control KSHV peptide does not block p65-Ser276 phosphorylation. A similar in vitro kinase assay was performed as in panel A except that the E1A-12 peptide was replaced with a peptide corresponding to the N terminus (residues 1 to 22) of the KSHV processivity factor PF8. (C) E1A-12 pep1-40 peptide does not alter phosphorylation of CREB in the PKAc assay. Increasing amounts (0, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 μM) of biotinylated CREB peptide that contains a PKA phosphorylation site were incubated with PKAc and ATP in the presence of different amounts (0, 20, or 50 μM) of E1A-12 pep1-40 peptide. The CREB peptide was then bound to streptavidin-coated plates. ELISA was performed using a phospho-PKA substrate [RRX(S/T)] antibody to detect CREB phosphorylation, and the absorbance was measured at 405 nm. Data represent averages of the results from three independent experiments. Error bars indicate standard deviations.

Junfang Jiao, et al. J Virol. 2010 August;84(15):7668-7674.

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