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1.
FIG. 7.

FIG. 7. From: Characterization of Functional Hepatitis C Virus Envelope Glycoproteins.

Low-pH treatment dissociates E1E2 complexes. 293T cells transfected to produce HCVpp were labeled for 24 h. Supernatants, either left untreated or exposed to a low pH, were immunoprecipitated with the anti-E2 MAb H50. Immunoprecipitates were analyzed by SDS-PAGE.

Anne Op De Beeck, et al. J Virol. 2004 March;78(6):2994-3002.
2.
FIG. 3.

FIG. 3. From: Characterization of Functional Hepatitis C Virus Envelope Glycoproteins.

Detection of the N termini of HCV envelope glycoproteins. HCVpp purified on a 20-to-60% sucrose gradient and lysates from cells expressing E1E2 were analyzed by Western blotting with MAbs recognizing an epitope located at the N terminus of E1 (MAb A4, recognizing amino acids 197 to 207) or E2 (MAb 9/27, recognizing amino acids 396 to 407). The anti-E2 MAb 3/11 was used as a control.

Anne Op De Beeck, et al. J Virol. 2004 March;78(6):2994-3002.
3.
FIG. 8.

FIG. 8. From: Characterization of Functional Hepatitis C Virus Envelope Glycoproteins.

E1E2 heterodimers associated with HCVpp interact with CD81. Supernatants (HCVpp) and cell lysates of 293T cells transfected to produce HCVpp were pulled down with a recombinant fusion protein containing the large extracellular loop of human CD81 (hCD81) or murine CD81 (mCD81) fused to glutathione S-transferase preadsorbed onto glutathione-Sepharose 4B beads. Precipitates were separated by SDS-PAGE followed by Western blot analysis with an anti-E1 (A4) or anti-E2 (3/11) MAb.

Anne Op De Beeck, et al. J Virol. 2004 March;78(6):2994-3002.
4.
FIG. 4.

FIG. 4. From: Characterization of Functional Hepatitis C Virus Envelope Glycoproteins.

Detection of the C termini of HCV envelope glycoproteins. (A) The envelope glycoproteins associated with HCVpp generated with either E1 plus E2, E1HA plus E2, or E1 plus E2HA or without envelope glycoproteins (control [C]) were analyzed by immunoprecipitation with MAb H53 followed by Western blotting with the anti-HA MAb 3F10, the anti-E1 MAb A4, or the anti-E2 MAb H52. Immunoglobulin light (IgL) and heavy (IgH) chains and SDS-resistant E1 oligomers (X) are indicated. (B) Infectivity of HCVpp containing HCV envelope proteins tagged at their C termini with an HA tag was evaluated by using the luciferase reporter gene. The infectivity of HCVpp containing E1HA plus E2 or E1 plus E2HA was compared to that of HCVpp containing wild-type HCV envelope glycoproteins (E1 plus E2).

Anne Op De Beeck, et al. J Virol. 2004 March;78(6):2994-3002.
5.
FIG. 6.

FIG. 6. From: Characterization of Functional Hepatitis C Virus Envelope Glycoproteins.

Neutralization of HCVpp by human MAbs. HCVpp were preincubated, before infection of Huh-7 cells, with saturating concentrations (20 μg/ml)of human anti-E2 MAbs. The saturating concentrations were determined by analyzing the extent of neutralization of HCVpp with several concentrations of several neutralizing antibodies (data not shown). A negative-control experiment using a nonspecific human MAb (RO4) was performed. Control pseudotype particles produced in the absence of envelope proteins were used to establish the background level, which was always below 1% (data not shown). Pseudotype particles bearing VSV-G protein were used in a control neutralization experiment. No neutralization response was observed with these control particles (data not shown). Results, determined by measuring the luciferase activity, are expressed as percentages of neutralization.

Anne Op De Beeck, et al. J Virol. 2004 March;78(6):2994-3002.
6.
FIG. 1.

FIG. 1. From: Characterization of Functional Hepatitis C Virus Envelope Glycoproteins.

Noncovalent E1E2 heterodimers are incorporated into HCVpp. At 16 h posttransfection, 293T cells transfected to produce HCVpp were metabolically labeled for 24 h. The supernatant (HCVpp) and cell lysate were immunoprecipitated with anti-E2 MAbs (H35, H48, and H53). The immunoprecipitates were analyzed by SDS-PAGE under reducing (R) or nonreducing (NR) conditions. Sizes of protein molecular mass markers are given on the left (in kilodaltons). HCV envelope proteins E1 and E2 as well as aggregates (Agg) and SDS-resistant E1E2 complexes are indicated on the right.

Anne Op De Beeck, et al. J Virol. 2004 March;78(6):2994-3002.
7.
FIG. 5.

FIG. 5. From: Characterization of Functional Hepatitis C Virus Envelope Glycoproteins.

Epitope exposure on HCV glycoproteins associated with HCVpp. 293T cells transfected to produce HCVpp were labeled for 24 h. Supernatants (HCVpp) and cell lysates were immunoprecipitated with anti-E2 MAbs. In some experiments, supernatants were treated with MES (pH 5.5) for 20 min at 37°C (HCVpp-low pH) and pH was neutralized before immunoprecipitation. The intensities of immunoprecipitated E2 were determined by phosphorimaging. MAbs are grouped as conformation dependent or conformation independent. The epitopes of the following antibodies have been identified: 6/16 (amino acids 384 to 395), 9/86a (amino acids 384 to 407), 3/11 (amino acids 412 to 423), 1/39 (amino acids 432 to 443), H47 (amino acids 448 to 463), 6/53 (amino acids 544 to 551), and H52 (amino acids 644 to 655). Although it recognizes HVR1, MAb 9/86a (asterisked at the bottom) is conformation dependent. H14 (Agg) recognizes aggregates of HCV envelope glycoproteins.

Anne Op De Beeck, et al. J Virol. 2004 March;78(6):2994-3002.
8.
FIG. 2.

FIG. 2. From: Characterization of Functional Hepatitis C Virus Envelope Glycoproteins.

Analyses of the glycans associated with HCV envelope proteins. (A) The glycans associated with HCV envelope glycoproteins are modified by Golgi enzymes. 293T cells transfected to produce HCVpp were labeled for 24 h. Supernatants (HCVpp) and cell lysates were immunoprecipitated with the anti-E2 MAb H53. The immunoprecipitates were either left untreated or treated with endo H or PNGase F and analyzed by SDS-PAGE. Deglycosylated forms of E1 and E2 are indicated by an asterisk. The endo H-resistant form of E1 is indicated by two asterisks. Sizes of protein molecular mass markers are given on the left (in kilodaltons). (B) The bulk of HCV envelope glycoproteins expressed in 293T cells is not modified by Golgi enzymes. 293T cells transfected for 24 h were pulse-labeled for 30 min and chased for different times as indicated (in hours). Cell lysates were immunoprecipitated with the anti-E2 MAb H53. The immunoprecipitates were either left untreated or treated with endo H and analyzed by SDS-PAGE. HCV envelope proteins E1 and E2 are indicated on the right. Deglycosylated forms of E1 and E2 are indicated by an asterisk.

Anne Op De Beeck, et al. J Virol. 2004 March;78(6):2994-3002.

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