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1.
Fig. 6.

Fig. 6. From: ?5?1-Integrin controls ebolavirus entry by regulating endosomal cathepsins.

Pretreatment of VSV-GPΔ to form VSV-GP19K rescues infection in the α5β1-integrin-negative CHO cells. VSV-GPΔ was either mock-treated (M; VSV-GPΔmock) or pretreated with thermolysin T to generate VSV-GP19k and analyzed by immunoblotting for GP1 (A) or used to infect the panel of CHO cells (B). Results shown in B are the averages from 9 experiments. Error bars indicate SEM. *, P ≤ 0.007 relative to CHO K1 cells. ⋀, P ≤ 0.01 relative to CHO B2 cells. **, P ≤ 0.04 relative to mock-treated VSV-GPΔ in the same cells.

Kathryn L. Schornberg, et al. Proc Natl Acad Sci U S A. 2009 May 12;106(19):8003-8008.
2.
Fig. 5.

Fig. 5. From: ?5?1-Integrin controls ebolavirus entry by regulating endosomal cathepsins.

Expression of α5β1-integrin enhances EboV GP-mediated infection of GD25 cells. (A) GD25 (GD) and β1GD25 (β1) cells were stained with Abs specific for α5- and β1-integrins, and the percentage of positive cells was measured by flow cytometry. (B) GD25 and β1GD25 cells were infected with VSV-GPΔ or VSV-G, and the percentage of infected cells was analyzed by flow cytometry. Data shown are the averages from 13 experiments. Error bars indicate SEM. *, P ≤ 0.03 relative to GD25 cells. (C) GD25 and β1GD25 cells were either untransfected or transfected with a plasmid encoding human CatL, and lysates were analyzed by immunoblotting for CatL.

Kathryn L. Schornberg, et al. Proc Natl Acad Sci U S A. 2009 May 12;106(19):8003-8008.
3.
Fig. 2.

Fig. 2. From: ?5?1-Integrin controls ebolavirus entry by regulating endosomal cathepsins.

Knockdown of α5-integrin in HeLa cells reduces EboV GP-mediated infection. HeLa cells transfected with a nontargeting control siRNA oligonucleotide (Con) or siRNA oligonucleotides targeting α5- or β1-integrin were biotinylated and lysed for avidin precipitation and immunoblotting (A) or infected with VSV-GPΔ or VSV-G (B). (A) Representative immunoblots, presented as in Fig. 1, with densitometry values presented below showing the amount of protein, normalized for GAPDH loading, relative to the control siRNA-treated cells. (B) Data shown are the averages from 5 experiments. Error bars indicate SEM. *, P ≤ 0.0008 relative to control siRNA-treated cells.

Kathryn L. Schornberg, et al. Proc Natl Acad Sci U S A. 2009 May 12;106(19):8003-8008.
4.
Fig. 1.

Fig. 1. From: ?5?1-Integrin controls ebolavirus entry by regulating endosomal cathepsins.

Expression of α5β1-integrin enhances EboV GP-mediated infection of CHO cells. (A) Total lysates and surface proteins of CHO K1 (K1), CHO B2 (B2), and CHO B2-α55) cells were immunoblotted for α5 (Upper) and β1 (Lower) integrin. Panels shown are from the same blot and same exposure; gaps indicate where lanes were removed. (B) The 3 CHO cell lines were infected with VSV-GP, VSV-GPΔ, or VSV-G, and the percentage of infected cells expressing GFP was measured by flow cytometry. Data shown are the averages from 10 experiments. Error bars indicate SEM. *, P ≤ 0.02 relative to CHO K1 cells. ⋀, P ≤ 0.05 relative to CHO B2 cells.

Kathryn L. Schornberg, et al. Proc Natl Acad Sci U S A. 2009 May 12;106(19):8003-8008.
5.
Fig. 4.

Fig. 4. From: ?5?1-Integrin controls ebolavirus entry by regulating endosomal cathepsins.

CatL activity levels and expression of the DC forms of CatB and CatL are reduced in the α5β1-negative CHO cells. CatL (A) and CatB (B) activity in cell lysates was measured against small fluorogenic peptide substrates. Results shown are the averages of 3 experiments. Error bars indicate SEM. *, P ≤ 0.001 relative to CHO K1 cells. ⋀, P ≤ 0.02 relative to CHO B2 cells. (C) Untransfected CHO cells and CHO cells transfected with a plasmid encoding human CatL were analyzed by immunoblotting for CatL. (D and E) Cell lysates and supernatants from equivalent numbers of cells transfected with plasmids encoding human CatB (D) or CatL (E) were concentrated and analyzed by immunoblotting for CatB or CatL as indicated. haDC, DC form of endogenous hamster CatL; huPro, huSC, and huDC, proform, SC form, and DC form, respectively, of overexpressed human CatB and CatL.

Kathryn L. Schornberg, et al. Proc Natl Acad Sci U S A. 2009 May 12;106(19):8003-8008.
6.
Fig. 3.

Fig. 3. From: ?5?1-Integrin controls ebolavirus entry by regulating endosomal cathepsins.

α5β1-Integrin regulates EboV GP-mediated entry after binding and after internalization. (A) VSV-GPΔ was bound to the panel of CHO cells at 4 °C, and the cells were either lysed directly (binding), treated with proteinase K to strip off bound virus and then lysed (no warm-up), or incubated at 37 °C for 2 h and then treated with proteinase K before lysing (internalization). Lysates were immunoblotted for the matrix protein of VSV (M). A representative blot from 8 separate experiments, presented as in Fig. 1, is shown, with densitometry values representing the amount of M protein, normalized for actin loading, relative to the parental CHO K1 cells. (B) VSV-GPΔ was bound to cells and internalized as described in Methods. Cells were permeabilized and stained with anti-EboV GP antibody (green) and phalloidin (red). Arrows indicate pseudovirions. See Fig. S1 A and B for larger version and quantitation. (C) Increasing concentrations of recombinant EboV RBR or control rabbit Fc (Con) were bound to cells, stained with AlexaFluor 488-conjugated Protein A, and analyzed by flow cytometry for the percentage of cells bound (Left) and for the mean fluorescence intensity (MFI) of cells (Right). (D) HIV-GPΔ and HIV-G were added to the panel of CHO cells, and fusion was measured as the percentage of blue cells. Results shown are the averages of normalized data from 8 experiments. The average percent infection in the CHO K1 cells was 26% for HIV-GPΔ and 64% for HIV-G. Error bars indicate SEM. *, P ≤ 6 × 10−10 relative to CHO K1 cells. ⋀, P ≤ 0.02 relative to CHO B2 cells.

Kathryn L. Schornberg, et al. Proc Natl Acad Sci U S A. 2009 May 12;106(19):8003-8008.

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