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1.
FIG. 2.

FIG. 2. From: Varicella-Zoster Virus Infection of Human Fibroblast Cells Activates the c-Jun N-Terminal Kinase Pathway .

Time course of JNK activation. Lysates of VZV- and mock-infected cells were collected at 2 h postinfection (day zero), day 1, day 2, day 3, and day 4 and were analyzed by immunoblotting. Blots were probed for phospho-JNK (P-JNK) and VZV antigen. Anisomycin-treated cells were used as a positive control. β-Actin levels were used as a loading control. M, mock infected; V, VZV infected; A+, anisomycin-treated positive control.

Heidi J. Zapata, et al. J Virol. 2007 January;81(2):977-990.
2.
FIG. 1.

FIG. 1. From: Varicella-Zoster Virus Infection of Human Fibroblast Cells Activates the c-Jun N-Terminal Kinase Pathway .

The JNK pathway is induced by VZV. HFF cells were either mock infected or infected with VZV for 3 to 4 days. Cells treated with anisomycin (250 ng/ml for 30 to 40 min) were used as a positive control for induction of the JNK pathway. Lysates were then collected and used for immunoblotting. The results are representative of more than three experiments. β-Actin levels were used as a loading control. M, mock infected; V, VZV infected; A, anisomycin treated; P, phospho; T, total.

Heidi J. Zapata, et al. J Virol. 2007 January;81(2):977-990.
3.
FIG. 4.

FIG. 4. From: Varicella-Zoster Virus Infection of Human Fibroblast Cells Activates the c-Jun N-Terminal Kinase Pathway .

JNK is more active in VZV-infected cells. Mock-treated, VZV-treated, and anisomycin-treated lysates were incubated with c-Jun beads (c-Jun-GST attached to glutathione beads) in order to pull down JNK. [γ-32P]ATP was added, and phosphorylation of c-Jun was used as a measure of kinase activity. Shown is an autoradiograph of c-Jun phosphorylation. Mock-, VZV-, and anisomycin-treated lysates were also incubated with glutathione beads as a negative control (also shown as an autoradiograph). Kinase assays were performed either with or without SP600125 (20 μM). Western blotting (WB) was performed to determine the presence of JNK in the pulldowns. M, mock infected; V, VZV infected; A, anisomycin treated; t, total.

Heidi J. Zapata, et al. J Virol. 2007 January;81(2):977-990.
4.
FIG. 5.

FIG. 5. From: Varicella-Zoster Virus Infection of Human Fibroblast Cells Activates the c-Jun N-Terminal Kinase Pathway .

Phospho-JNK and phospho-c-Jun accumulate in the nuclei of VZV-infected cells. (A through E) Localization of phospho-JNK. Anisomycin-treated cells were used as a positive control. Confluent cells were either mock infected or VZV infected for 24 h on glass slides. Slides were fixed and probed for phospho-JNK and VZV glycoproteins. Proteins were visualized using immunofluorescent confocal microscopy. (A) Phospho-JNK localizes to the nuclei of VZV-infected cells. (B) Enlargement of boxed area in panel A. The smaller panel on the right shows an enlargement of the red channel alone. (C) Anisomycin-treated cells, used as a positive control. (D) Mock-treated cells. (E) Antibody control with VZV-infected cells. (F through I) Localization of phospho-c-Jun. (F) Phospho-c-Jun localizes to the nuclei of VZV-infected cells. (G) Anisomycin-treated cells, used as a positive control. (H) Mock-treated cells. (I) Antibody control with VZV-infected cells. Red, phospho-JNK or phospho-c-Jun; green, VZV glycoproteins; blue, nuclei. Magnification, ×200.

Heidi J. Zapata, et al. J Virol. 2007 January;81(2):977-990.
5.
FIG. 3.

FIG. 3. From: Varicella-Zoster Virus Infection of Human Fibroblast Cells Activates the c-Jun N-Terminal Kinase Pathway .

Effects of SP600125 on VZV replication. (A) Dose dependence of SP600125. HFF cells were infected with VZV and treated either with increasing amounts of SP600126 (0 to 20 μM) or with diluent alone. The yield of VZV was determined by a plaque assay. (B) Specificity of SP600125. Lysates of VZV-infected cells, treated with increasing amounts of SP600126, were analyzed by immunoblotting and probed for phospho-c-Jun (P-cJun), c-Jun, and VZV antigen. A+, anisomycin-treated positive control; Ø, no inhibitor added. (C) Plaque reduction assay. Confluent HFF cells were either mock infected or VZV infected on glass slides for 48 h, with or without 20 μM SP600125. Control cells were treated with an equivalent amount of diluent (DMSO). Slides were analyzed by immunofluorescent confocal microscopy. (D) The cytotoxicity of SP600125 was determined by a neutral red uptake assay. NT, no treatment.

Heidi J. Zapata, et al. J Virol. 2007 January;81(2):977-990.
6.
FIG. 6.

FIG. 6. From: Varicella-Zoster Virus Infection of Human Fibroblast Cells Activates the c-Jun N-Terminal Kinase Pathway .

Phospho-JNK accumulates in the nuclei of infected cells at early times. Confluent cells were either mock infected or VZV infected for 24 h on glass slides in the absence of PAA (A to D) or with PAA (E to K). Anisomycin-treated cells were used as a positive control. Slides were fixed and probed for phospho-JNK and IE62. Proteins were visualized using immunofluorescent confocal microscopy. (A) VZV-infected cells in the absence of PAA. Thin arrows indicate late infected cells, with cytoplasmic IE62 and nuclear phospho-JNK. Thick arrows indicate early infected cells, where IE62 is entirely nuclear and phospho-JNK is present in the nucleus. (B) Anisomycin-treated cells. (C) Mock-treated cells. (D) Antibody control with VZV-infected cells. (E) VZV-infected cells in the presence of PAA. Areas of colocalization between phospho-JNK and nuclear IE62 are boxed. (F) Anisomycin-treated cells. (G) Mock-treated cells. (H) Antibody control with VZV-infected cells. For panels A through H, IE62 stained red, phospho-JNK stained green, and nuclei stained blue. (I) Confluent cells were infected with VZV for 24 h on glass slides in the presence of PAA. Slides were fixed and probed for phospho-JNK (red), VZV glycoproteins (green), and nuclei (blue). (J) Red channel. Red, phospho-JNK. (K) Enlargement of an area of panel J. Magnification, ×200 (A to H); magnification, ×400 (I to K).

Heidi J. Zapata, et al. J Virol. 2007 January;81(2):977-990.
7.
FIG. 7.

FIG. 7. From: Varicella-Zoster Virus Infection of Human Fibroblast Cells Activates the c-Jun N-Terminal Kinase Pathway .

JNK is incorporated into VZV virions. (A) Negative stain of purified VZV virions. VZV particles were negatively stained with 1% uranyl acetate and viewed by TEM (magnification, ×49,000). The top arrow indicates an intact virion with a visible nucleocapsid. The bottom arrow indicates a disrupted virion with the nucleocapsid and tegument spilling out of the envelope. (B) Purified virions were analyzed by immunoblotting and probed with antibodies to the proteins indicated. V, VZV-infected cell lysate; M, mock-infected cell lysate; VP, purified virion particles. (C) Virion detergent and protease treatments. Purified virions were treated with 1% Triton X-100 for 30 min and separated into two fractions: the pelleted virus particles (lane 1) and the supernatant (lane 2). Purified virions were also treated with trypsin alone for 15 min (lane 3) and with trypsin and 1% Triton X-100 for 5 (lane 4) or 15 (lane 5) min. Treated virions were analyzed by immunoblotting and probed with appropriate antibodies. VP, untreated virus particles. (D) Immunoelectron microscopy. Heavily infected cells were sectioned, probed with a polyclonal anti-JNK antibody and a secondary antibody conjugated to 10-nm-diameter gold beads, and viewed by TEM. Magnification: ×40,000 for the top panel and ×10,000 for the bottom panel.

Heidi J. Zapata, et al. J Virol. 2007 January;81(2):977-990.

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