Journal: Viruses

Article Title: Recombinant Human Cytomegalovirus Expressing an Analog-Sensitive Kinase pUL97 as Novel Tool for Functional Analyses

doi: 10.3390/v14102285

Figure Lengend Snippet: Replication kinetics of BADwt and BAD-UL97-as1 after treatment with 3MB-PP1. Cells were treated with either 40 µM of 3MB-PP1 or with DMSO one day before infection. The next day, cells were infected with BADwt or BAD-UL97-as1 with 4 genomes/cell. Samples were taken and viral DNA was extracted from infected cells and the cell supernatant at the indicated time points. The intracellular and extracellular viral genome copies were quantified by quantitative PCR analysis.

Article Snippet: 3MB-PP1 (Calbiochem Merck, 529582-5MG) and maribavir (MBV; MedChem Express, HY-16305) were added to the cell culture in a concentration of 40 µM for 3MB-PP1 or 20 µM for MBV once up to 18 h before infection or at different time points during infection.

Techniques: Infection, Real-time Polymerase Chain Reaction

Journal: Viruses

Article Title: Recombinant Human Cytomegalovirus Expressing an Analog-Sensitive Kinase pUL97 as Novel Tool for Functional Analyses

doi: 10.3390/v14102285

Figure Lengend Snippet: Phosphorylation of the retinoblastoma protein (Rb). Cells were pre-treated with DMSO, 3MB-PP1 or MBV one day before infection. The next day, cells were infected with BADwt or BAD-UL97-as1. Cells were harvested and lysed at 5 d.p.i. and analyzed in an SDS-PAGE and Western blot using a phospho-specific antibody for residues 807/811 of Rb. Antibodies against full protein Rb, the viral proteins pp28 and pUL97 were probed for reference.

Article Snippet: 3MB-PP1 (Calbiochem Merck, 529582-5MG) and maribavir (MBV; MedChem Express, HY-16305) were added to the cell culture in a concentration of 40 µM for 3MB-PP1 or 20 µM for MBV once up to 18 h before infection or at different time points during infection.

Techniques: Infection, SDS Page, Western Blot

Journal: Viruses

Article Title: Recombinant Human Cytomegalovirus Expressing an Analog-Sensitive Kinase pUL97 as Novel Tool for Functional Analyses

doi: 10.3390/v14102285

Figure Lengend Snippet: ( A ) BAD-UL97-as1 infected cells (10 genomes/cell) were harvested at 5 d.p.i. 40 µM of 3MB-PP1 was added at either 5 days, 2 days, 1 day or 4 h before harvest. The samples were analyzed by SDS-Page and Western blot analysis, using a specific antibody against the phosphosite 807/811 of Rb. The quantification was performed by measuring the protein intensity of pRb and Rb using Image Studio Lite Version 5.2.5. The ratios (pRb807/811/Rb) in dependence of 3MB-PP1 are shown. Thereby the corresponding DMSO sample was indicated as 1 (n = 2). ( B ) BAD-UL97-as1 (2 genomes/cell) infected HFFs were treated with 3MB-PP1 at different time points during infection. At 5 days, 2 days, 1 day or 4 h before harvest, the inhibitor was added. After 5 days post-infection, the supernatant was collected, DNA was isolated and the viral DNA of three technical replicates was quantified by TaqMan-PCR. The means ± standard deviations of each sample are shown.

Article Snippet: 3MB-PP1 (Calbiochem Merck, 529582-5MG) and maribavir (MBV; MedChem Express, HY-16305) were added to the cell culture in a concentration of 40 µM for 3MB-PP1 or 20 µM for MBV once up to 18 h before infection or at different time points during infection.

Techniques: Infection, SDS Page, Western Blot, Isolation

Journal: Viruses

Article Title: Recombinant Human Cytomegalovirus Expressing an Analog-Sensitive Kinase pUL97 as Novel Tool for Functional Analyses

doi: 10.3390/v14102285

Figure Lengend Snippet: Dysregulated protein groups after 3MB-PP1 treatment in uninfected HFF. Pathway and process enrichment analysis of proteins up-regulated after 3MB-PP1 treatment (log2 ≥ 0.7 and p -value ≤ 0.05) ( A ). The top 20 clusters with their representative enriched terms are plotted according to their p -value in log base 10 (Log10 (P)). Enrichment analysis was performed with Metascape . Pathway and process enrichment analysis of proteins down-regulated after 3MB-PP1 treatment (log2 ≤ −0.7 and p -value ≤ 0.05) ( B ). The top 20 clusters with their representative enriched terms are plotted according to their p -value in log base 10 (Log10 (P)). Enrichment analysis was performed with Metascape . Pathway results are shown with the number of proteins found in the dataset and computed FDR for pathway enrichment (FDR < 0.001).

Article Snippet: 3MB-PP1 (Calbiochem Merck, 529582-5MG) and maribavir (MBV; MedChem Express, HY-16305) were added to the cell culture in a concentration of 40 µM for 3MB-PP1 or 20 µM for MBV once up to 18 h before infection or at different time points during infection.

Techniques:

Journal: Viruses

Article Title: Recombinant Human Cytomegalovirus Expressing an Analog-Sensitive Kinase pUL97 as Novel Tool for Functional Analyses

doi: 10.3390/v14102285

Figure Lengend Snippet: HFFs were cultivated in T175 flasks and used for infection with parental HCMV BADwt or recombinant BAD-UL97-as1 at a MOI of 0.5 for 4 d. An optional treatment with 40 µM of 3MB-PP1 (+, 3MB-PP1; −, DMSO solvent controls) was performed starting 4 h prior to sample collection and again during cell lysis. Total cell lysates were prepared and used for cyclin B1-specific CoIP with the indicated antibodies (see black boxes; Fc fragment was used as a negative control) under the continuous presence of 3MB. ( A , B ) show two independently produced biological replicates of this CoIP experiment, in order to illustrate the range of variability of individual signal strengths of detected protein bands. The CoIP samples were subjected to SDS-PAGE/Wb analysis (CoIP) and additional control stainings were performed to verify successful immunoprecipitation (cyclin IP control) and protein expression levels (lysate controls).

Article Snippet: 3MB-PP1 (Calbiochem Merck, 529582-5MG) and maribavir (MBV; MedChem Express, HY-16305) were added to the cell culture in a concentration of 40 µM for 3MB-PP1 or 20 µM for MBV once up to 18 h before infection or at different time points during infection.

Techniques: Infection, Recombinant, Lysis, Negative Control, Produced, SDS Page, Immunoprecipitation, Expressing