Journal: Retrovirology

Article Title: HIV-1 Vif N-terminal Motif is required for recruitment of Cul5 to Suppress APOBEC3

doi: 10.1186/1742-4690-11-4

Figure Lengend Snippet: Vif N-terminal amino acids are responsible for Cul5 interaction, in vitro . Vif wild-type and single alanine mutants were co-expressed with N-terminal Cul5, N-terminal 6X-His-CBF-β (residues 1–140), and Elo B/C. Next, the complex was pulled down using nickel affinity purification. A) While Vif wild-type and H28A mutant pull down Cul5 efficiently, H27A, M29A and Y30A mutants are unable to bind Cul5 efficiently. B) Quantitative measurement of the Cul5 band intensity was performed indicating the relative amount of Cul5 bound to Vif wild-type and mutant protein complexes. FL – full length, N-terminal – amino-terminus, 6X-His – 6X histidine tag.

Article Snippet: Wild-type or mutant Vif was co-expressed with Cul5, Elo B/C, and 6X-His-tagged CBF-β (amino acids 1–140) in NiCo21(DE3) competent E. coli (NEB) at 23C with 0.2 mM IPTG.

Techniques: In Vitro, Affinity Purification, Mutagenesis

Journal: Retrovirology

Article Title: HIV-1 Vif N-terminal Motif is required for recruitment of Cul5 to Suppress APOBEC3

doi: 10.1186/1742-4690-11-4

Figure Lengend Snippet: Select Vif N-terminal mutants have a reduced ability to bind Cul5 in mammalian cells. HA-tagged Vif wild-type and mutant proteins along with CBF-β were overexpressed in HEK 293 T cells. Two days post-transfection, cells were lysed and cleared lysate was mixed with anti-HA matrix affinity beads for 4-8 hrs. Incubated beads were washed several times followed by elution of bound proteins. Select Vif N-terminal mutants (V25A, H27A, M29A, and Y30A) that do not efficiently degrade A3G and A3F have a reduced ability to co-precipitate Cul5; however, CBF-β and Elo B/C can still bind Vif.

Article Snippet: Wild-type or mutant Vif was co-expressed with Cul5, Elo B/C, and 6X-His-tagged CBF-β (amino acids 1–140) in NiCo21(DE3) competent E. coli (NEB) at 23C with 0.2 mM IPTG.

Techniques: Mutagenesis, Transfection, Incubation

Journal: Retrovirology

Article Title: HIV-1 Vif N-terminal Motif is required for recruitment of Cul5 to Suppress APOBEC3

doi: 10.1186/1742-4690-11-4

Figure Lengend Snippet: Vif N-terminal mutants localize to cytoplasm, but are inefficient at restoring HIV infectivity. HA-tagged Vif wild-type and mutant proteins along with CBF-β were overexpressed in HEK 293 T cells. Two days post-transfection, cells were lysed and cleared lysate was mixed with anti-HA matrix affinity beads for 4-8 hrs. Incubated beads were washed several times followed by elution of bound proteins. A) Select Vif N-terminal mutants that do not efficiently degrade A3G and A3F have a reduced ability to co-precipitate Cul5; however, CBF-β and Elo B/C can still bind Vif. B) Plasmids (Vif-YFP 2 ug and CBF-β 0.5 ug) were transfected into 293 T cells using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol. Cells were visualized at 25°C using a Zeiss LSM510-Meta confocal imaging system. Imaging demonstrates that the Vif double mutant V25/H27A and single mutant H108A localize to the cytoplasm of the cell similar to wild-type. C) Vif wild-type and mutant containing virus were produced and used to infect MAGI cells. Infected cells were stained using X-gal. The histogram demonstrates that Cul5-binding deficient Vif mutants were inefficient at restoring HIV infectivity in the presence of A3G. Error bars represent the standard error from triplicate experiments. Capsid p24 levels are shown in the western blot.

Article Snippet: Wild-type or mutant Vif was co-expressed with Cul5, Elo B/C, and 6X-His-tagged CBF-β (amino acids 1–140) in NiCo21(DE3) competent E. coli (NEB) at 23C with 0.2 mM IPTG.

Techniques: Infection, Mutagenesis, Transfection, Incubation, Imaging, Produced, Staining, Binding Assay, Western Blot

Journal: Retrovirology

Article Title: HIV-1 Vif N-terminal Motif is required for recruitment of Cul5 to Suppress APOBEC3

doi: 10.1186/1742-4690-11-4

Figure Lengend Snippet: CD spectroscopy analysis reveals that Vif mutant complexes are structurally different from wild-type. Purified Vif complexes including 6X-His-CBF-β (residues 1–182) and Elo B/C were purified by nickel affinity and size exclusion chromatography. Each complex was analyzed by circular dichroism spectroscopy. A) CD spectra for Vif wild-type and mutant complexes showed a distinction in the minima at 208 and 222 for mutant complexes that do not bind Cul5, suggesting these mutants have more alpha helical structure. B) Spectra analysis reveals differences between wild-type and mutant complex secondary structure and confirms that the mutants that do not bind Cul5 have a higher percentage of alpha helical structures; however, the percentage of beta-sheet structures is reduced.

Article Snippet: Wild-type or mutant Vif was co-expressed with Cul5, Elo B/C, and 6X-His-tagged CBF-β (amino acids 1–140) in NiCo21(DE3) competent E. coli (NEB) at 23C with 0.2 mM IPTG.

Techniques: Spectroscopy, Mutagenesis, Purification, Size-exclusion Chromatography

Journal: ACS chemical neuroscience

Article Title: Passage Variation of PC12 Cells Results in Inconsistent Susceptibility to Externally Induced Apoptosis

doi: 10.1021/acschemneuro.6b00208

Figure Lengend Snippet: Optimization and validation of etoposide-induced cell death in PC12 cells (passage 9–13). 50 × 103 cells/well. (a) Approximately 50% cell death was observed after 48 h. Four replicates per experiment. Values are represented as mean ± SD. Multiple t test, Holm-Sidak method, alpha = 5.000%, ****p < 0.0001. (b) Fluorescence measured after 48 h. Flupirtine at 3 μM concentration rescued the cells from apoptosis. Experiment repeated thrice. Values are represented as mean ± SD. Unpaired two-tailed t test, 95% CI, ****p < 0.0001 in comparison to vehicle. ####p < 0.0001 in comparison to etoposide treatment.

Article Snippet: 5 Etoposide (Chem-Impex International, 28435) was stored as a working stock solution of 150 μ g/mL at −20 °C for up to 3 months.

Techniques: Fluorescence, Concentration Assay, Two Tailed Test, Comparison

Journal: ACS chemical neuroscience

Article Title: Passage Variation of PC12 Cells Results in Inconsistent Susceptibility to Externally Induced Apoptosis

doi: 10.1021/acschemneuro.6b00208

Figure Lengend Snippet: NGF differentiated PC12 cells show a statistically significant difference in sensitivity to apoptosis induced by etoposide between early and late passages at 72 h post insult. (a) Passage 10. Three replicates per experiment. Values are represented as mean ± SD. One-way ANOVA, Dunnett test, 95% CI. ****p < 0.0001. (b) Passage 17. Three replicates per experiment. Values are represented as mean ± SD. One-way ANOVA, Dunnett test, 95% CI. ****p < 0.0001. (c) Comparison of 72 h etoposide treatment in passage 10 and 17 PC12 cells differentiated using NGF. Unpaired two-tailed t test, 95% CI, ****p < 0.0001.

Article Snippet: 5 Etoposide (Chem-Impex International, 28435) was stored as a working stock solution of 150 μ g/mL at −20 °C for up to 3 months.

Techniques: Comparison, Two Tailed Test