Journal: Cells
Article Title: Globoside Is an Essential Intracellular Factor Required for Parvovirus B19 Endosomal Escape
doi: 10.3390/cells13151254
Figure Lengend Snippet: pH modulates the affinity of B19V for globoside and VP1uR. ( A ) Recombinant VP1u was allowed to interact with UT7/Epo cells at 4 °C for 1 h or at 37 °C for 30 min under neutral (pH 7.2) or acidic (pH 5.0) conditions. Cells were subsequently washed to remove unbound virus and fixed and stained for VP1u using an anti-Flag antibody. Where indicated, a trypsinization (tryp) step was performed to remove bound VP1u from the cell surface. ( B ) Recombinant VP1u was allowed to interact with UT7/Epo cells at 4 °C and neutral pH for 30 min followed by acidification (pH 5.0) and incubation for another 30 min. Cells were washed with ice-cold buffer at pH 5.0, fixed, and stained for VP1u using an anti-Flag antibody. ( C ) Binding affinity of B19V for globoside was determined by incubating purified B19V (3 × 10 9 ) with RBCs (0.5%) in 100 µL buffer at pH ranging from 7.2 to 5.6 at RT for 1 h. Cells were washed with incubation buffer and DNA was extracted and quantified by qPCR. The binding affinity of VP1u for VP1uR was quantified by incubating PP7-VP1u (10 10 ) with UT7/Epo cells in 100 µL buffer at pH ranging from 7.2 to 5.6 at 4 °C for 1 h. Cells were washed with incubation buffer and DNA was extracted and quantified by qPCR. Results are presented as the mean of two independent experiments ± standard deviation (SD).
Article Snippet: PP7 bacteriophages were obtained from ATCC, propagated in Pseudomonas aeruginosa , and modified as previously indicated [ ].
Techniques: Recombinant, Virus, Staining, Incubation, Binding Assay, Purification, Standard Deviation
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