Journal:

Article Title: Expression of Hepatitis C Virus Proteins Inhibits Signal Transduction through the Jak-STAT Pathway

doi:

Figure Lengend Snippet: Inhibition of IFN signaling in cells expressing HCV proteins. Results of UTA-6 cells (a U-2 OS-derived cell line transfected with the tTA) are shown in lanes 1 to 4, and of UHCV-11 and UHCV-32 cells (containing both tTA and the HCV cDNA) in lanes 5 to 8 and 9 to 12, respectively. The cells were cultured in the presence or absence of tetracycline, as indicated by Tet + (lanes 1, 2, 5, 6, 9, and 10) and Tet − (lanes 3, 4, 7, 8, 11, and 12). Cells were then either left untreated (IFN −, odd-numbered lanes) or stimulated with 500 U of IFN-α per ml for 30 min (IFN +, even-numbered lanes). (A) EMSA with nuclear extracts and the ISRE oligonucleotide probe. The position of ISGF3 is indicated by an arrow. In cells expressing HCV proteins, the induction of ISGF3 by IFN-α is inhibited (lanes 8 and 12). Antibodies to Stat1 (α1, lane 14) and Stat2 (α2, lane 15) interfere with ISGF3, resulting in the disappearance of the gel shift signals. Stat3 specific serum (α3, lane 16) has no effect. (B) The same extracts were tested with an m67 oligonucleotide probe. The position of SIF-A, SIF-B, and SIF-C are indicated. IFN-α-induced formation of Stat1 and Stat3 complexes is impaired in cells expressing viral proteins (lanes 8 and 12). Antiserum to Stat1 (α1, lane 14) supershifts SIF-B and SIF-C. Antiserum to Stat3 (α3, lane 16) supershifts SIF-A and SIF-B. (C) Western blot with MAb C7-50 against the HCV core protein with the corresponding cytoplasmic extracts. Viral proteins are expressed only in UHCV-11 and UHCV-32 cells that were cultured in the absence of tetracycline (lanes 7, 8, 11, and 12). Molecular mass markers in kilodaltons are indicated on the left. (D) UGFP-9 cells were cultured for 24 h in the presence (lanes 1 and 2) or absence (lanes 3 and 4) of tetracycline, then either left untreated (lanes 1 and 3) or stimulated for 30 min with 500 U of human IFN-α (lanes 2 and 4) per ml and subsequently processed for EMSA with the ISRE oligonucleotide probe. The position of ISGF3 is indicated by an arrow.

Article Snippet: Antibodies against Stat1 (sc417) and Stat2 (sc476) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.), antibodies against Stat3 (06-373) and phosphotyrosine (05-321) came from Upstate Biotechnology (Lake Placid, N.Y.), antibodies against ISGF3γ-p48 were from Transduction Labs (Lexington, Ky.), and antibodies against phosphorylated Stat1 were from New England Biolabs (Beverly, Mass.).

Techniques: Inhibition, Expressing, Derivative Assay, Transfection, Cell Culture, Electrophoretic Mobility Shift Assay, Western Blot

Journal:

Article Title: Expression of Hepatitis C Virus Proteins Inhibits Signal Transduction through the Jak-STAT Pathway

doi:

Figure Lengend Snippet: Stat3 activation by LIF is inhibited by HCV proteins. UTA-6 and UHCV-32 cells were cultured in the presence or absence of tetracycline (Tet + and −, respectively) and treated with LIF where indicated. (A) Gel shift assay with the m67 oligonucleotide probe shows induction of Stat3 shifts (arrow). The shift intensity is weaker in UHCV-32 cells expressing viral proteins. (B) The same gel was analyzed with a phosphorimager. Quantification of Stat3 signals reveals the inhibition of LIF-induced Stat3 shifts by viral protein expression. Arbitrary signal density units are shown on the left. (C) Western blot with the corresponding cytoplasmic extracts and the anti-HCV-core-specific MAb C7-50. Molecular mass markers in kilodaltons are indicated on the left.

Article Snippet: Antibodies against Stat1 (sc417) and Stat2 (sc476) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.), antibodies against Stat3 (06-373) and phosphotyrosine (05-321) came from Upstate Biotechnology (Lake Placid, N.Y.), antibodies against ISGF3γ-p48 were from Transduction Labs (Lexington, Ky.), and antibodies against phosphorylated Stat1 were from New England Biolabs (Beverly, Mass.).

Techniques: Activation Assay, Cell Culture, Electrophoretic Mobility Shift Assay, Expressing, Inhibition, Western Blot

Journal:

Article Title: Expression of Hepatitis C Virus Proteins Inhibits Signal Transduction through the Jak-STAT Pathway

doi:

Figure Lengend Snippet: Phosphorylation of STAT proteins is not impaired by HCV protein expression. UHCV-32 cells were cultured for 24 h in the presence or absence of tetracycline (Tet + or −) and then either were left untreated (−) or were stimulated with IFN-α (+). Whole-cell lysates were prepared and used for immunoprecipitations with anti-Stat1 specific serum. (A) The precipitated proteins were separated by sodium dodecyl sulfate–7% polyacrylamide gel electrophoresis, followed by Western blot analysis with antiserum to the phosphorylated form of Stat1 (α-Stat1-P), Stat1 in general (α-Stat1), and Stat2 (α-Stat2): Stat1 phosphorylation and heterodimerization with Stat2 were unaffected by HCV protein expression. (B) Supernatants of the immunoprecipitation pellets were analyzed by Western blot with antisera specific for Stat2 (α-Stat2), Stat3 (α-Stat3), and ISGF3γ-p48 (α-p48). The expression levels of these proteins were not influenced by viral protein expression. Molecular mass markers in kilodaltons are indicated on the left.

Article Snippet: Antibodies against Stat1 (sc417) and Stat2 (sc476) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.), antibodies against Stat3 (06-373) and phosphotyrosine (05-321) came from Upstate Biotechnology (Lake Placid, N.Y.), antibodies against ISGF3γ-p48 were from Transduction Labs (Lexington, Ky.), and antibodies against phosphorylated Stat1 were from New England Biolabs (Beverly, Mass.).

Techniques: Expressing, Cell Culture, Polyacrylamide Gel Electrophoresis, Western Blot, Immunoprecipitation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TLR3 activation augments matrix metalloproteinase production through reactive nitrogen species generation in human lung fibroblasts.

doi: 10.4049/jimmunol.1302919

Figure Lengend Snippet: FIGURE 9. Responsiveness of NHLFs and DHLF-As to poly(I:C). NHLFs (normal) (n = 3) and DHLF-As (asthma) (n = 4) were incubated with 30 mg/ml poly(I:C) or vehicle for 48 h. MMP-9 (A and B), -2 (A and C) and -1 (D) were analyzed by zymography. Fold increase was calculated by dividing the band intensity of MMPs in the poly(I:C)-treated group by that in the vehicle-treated group. The fold increases in the MMPs from NHLFs are compared with those from DHLF-As (B–D). Expression of TLR3 (E) and iNOS (F) in the cells was analyzed by immunoblotting. *p , 0.05 versus NHLFs. M, markers of MMPs.

Article Snippet: The cells were incubated with mouse monoclonal anti-TLR3 Ab (1:100 dilution; Imgenex) or mouse IgG as a negative control for detection of TLR3.

Techniques: Incubation, Zymography, Expressing, Western Blot