Journal:

Article Title: Human Immunodeficiency Virus Type 1 Replication Is Modulated by Host Cyclophilin A Expression Levels

doi:

Figure Lengend Snippet: Primary structure of the HIV-1 Gag polyprotein proline-rich loop that binds cyclophilin A and confers cyclophilin A dependence on HIV-1 replication. The numbers refer to amino acid residues with respect to the amino terminus of the Gag polyprotein. The asterisks indicate the positions of G221, P222, and A224, residues important to this study.

Article Snippet: Size-separated cellular proteins or virion-associated proteins were transferred to polyvinylidene difluoride membranes with a Bio-Rad (Hercules, Calif.) mini-blotting apparatus and probed with a rabbit anti-cyclophilin A antibody (Affinity BioReagents, Heshanic Station, N.J.) or with a murine monoclonal anti-HIV-1 capsid antibody (Dupont NEN).

Techniques:

Journal:

Article Title: Human Immunodeficiency Virus Type 1 Replication Is Modulated by Host Cyclophilin A Expression Levels

doi:

Figure Lengend Snippet: HIV-1 A224E mutant virus is cyclosporine resistant in Jurkat cells and cyclosporine dependent in H9 cells. Infection of the Jurkat T-cell line (A) or of the H9 T-cell line (B) was initiated by transfection of proviral DNA pNL4-3 encoding the A224E amino acid mutation. Cyclosporine (CsA) was maintained in the cell culture medium at the indicated concentrations. The accumulation of extracellular virions in the culture supernatant resulting from viral spread through the culture is indicated by the reverse transcriptase (RT) activity in the supernatant at the indicated times posttransfection.

Article Snippet: Size-separated cellular proteins or virion-associated proteins were transferred to polyvinylidene difluoride membranes with a Bio-Rad (Hercules, Calif.) mini-blotting apparatus and probed with a rabbit anti-cyclophilin A antibody (Affinity BioReagents, Heshanic Station, N.J.) or with a murine monoclonal anti-HIV-1 capsid antibody (Dupont NEN).

Techniques: Mutagenesis, Infection, Transfection, Cell Culture, Activity Assay

Journal:

Article Title: Human Immunodeficiency Virus Type 1 Replication Is Modulated by Host Cyclophilin A Expression Levels

doi:

Figure Lengend Snippet: P222A, a Gag mutation that confers reduced cyclophilin A affinity to the Gag polyprotein, rescues the replication of HIV-1 mutant A224E in H9 cells when the two mutations are expressed in cis. Jurkat T cells (A) or H9 T cells (B) were transfected with the wild-type proviral DNA pNL4-3 or with pNL4-3 encoding the P222A/A224E double mutation. RT, reverse transcriptase.

Article Snippet: Size-separated cellular proteins or virion-associated proteins were transferred to polyvinylidene difluoride membranes with a Bio-Rad (Hercules, Calif.) mini-blotting apparatus and probed with a rabbit anti-cyclophilin A antibody (Affinity BioReagents, Heshanic Station, N.J.) or with a murine monoclonal anti-HIV-1 capsid antibody (Dupont NEN).

Techniques: Mutagenesis, Transfection

Journal:

Article Title: Human Immunodeficiency Virus Type 1 Replication Is Modulated by Host Cyclophilin A Expression Levels

doi:

Figure Lengend Snippet: H9 cells express more cyclophilin A (CyPA) than do Jurkat cells. (A) Jurkat cells (lanes 1, 3, and 5) and H9 cells (lanes 2, 4, and 6 to 9) were grown to a density of 106/ml, lysed in radioimmunoprecipitation assay buffer, and processed for Western blotting with a primary anti-cyclophilin A antibody. Lanes 1 to 4, samples normalized by cell number. The quantity of soluble cell lysate loaded per lane, as measured by number of cell equivalents, is indicated above the lanes. Lanes 5 to 9, samples normalized by quantity of total protein in the soluble cell lysate. The numbers above the lanes indicate the amount of total protein loaded per well. (B) Replication of wild-type HIV-1 in Jurkat or H9 cells after initiation of infection with a common stock of virus produced by transfection of 293T cells with pNL4-3. RT, reverse transcriptase. (C) Virions were purified from the supernatants of the cultures used for panel B on day 8 postinfection. Virion-associated protein was probed in a Western blot with a primary anti-cyclophilin A antibody. Lanes 1 and 2, samples from Jurkat and H9 cell cultures, respectively, normalized for reverse transcriptase activity and capsid protein. Lanes 3 and 4, serial twofold dilutions of the H9 sample, as indicated.

Article Snippet: Size-separated cellular proteins or virion-associated proteins were transferred to polyvinylidene difluoride membranes with a Bio-Rad (Hercules, Calif.) mini-blotting apparatus and probed with a rabbit anti-cyclophilin A antibody (Affinity BioReagents, Heshanic Station, N.J.) or with a murine monoclonal anti-HIV-1 capsid antibody (Dupont NEN).

Techniques: Radio Immunoprecipitation, Western Blot, Infection, Produced, Transfection, Purification, Activity Assay

Journal:

Article Title: Human Immunodeficiency Virus Type 1 Replication Is Modulated by Host Cyclophilin A Expression Levels

doi:

Figure Lengend Snippet: HIV-1 proviruses expressing Gag mutants with attenuated cyclophilin A affinity cannot promote a spreading infection in Jurkat cells (A) but do replicate in H9 cells (B). Infection was initiated by direct transfection of wild-type or mutant proviral DNA pNL4-3 into the T-cell lines. RT, reverse transcriptase.

Article Snippet: Size-separated cellular proteins or virion-associated proteins were transferred to polyvinylidene difluoride membranes with a Bio-Rad (Hercules, Calif.) mini-blotting apparatus and probed with a rabbit anti-cyclophilin A antibody (Affinity BioReagents, Heshanic Station, N.J.) or with a murine monoclonal anti-HIV-1 capsid antibody (Dupont NEN).

Techniques: Expressing, Infection, Transfection, Mutagenesis

Journal:

Article Title: Human Immunodeficiency Virus Type 1 Replication Is Modulated by Host Cyclophilin A Expression Levels

doi:

Figure Lengend Snippet: HIV-1 replication in H9 cells is resistant to inhibition by cyclosporine. Infection of Jurkat T cells (A) or of H9 T cells (B) was initiated by transfection of wild-type (WT) proviral DNA pNL4-3. Cyclosporine (CsA) was maintained in the cell culture medium for the duration of the experiment at the indicated concentrations. RT, reverse transcriptase.

Article Snippet: Size-separated cellular proteins or virion-associated proteins were transferred to polyvinylidene difluoride membranes with a Bio-Rad (Hercules, Calif.) mini-blotting apparatus and probed with a rabbit anti-cyclophilin A antibody (Affinity BioReagents, Heshanic Station, N.J.) or with a murine monoclonal anti-HIV-1 capsid antibody (Dupont NEN).

Techniques: Inhibition, Infection, Transfection, Cell Culture

Journal:

Article Title: Human Immunodeficiency Virus Type 1 Replication Is Modulated by Host Cyclophilin A Expression Levels

doi:

Figure Lengend Snippet: HIV-1 Gag mutants expressed in cis do not rescue the replication of Vif-defective HIV-1 in H9 cells. Infection of H9 T cells was initiated by transfection of wild-type (WT) or mutant proviral DNA pNL4-3. RT, reverse transcriptase. The Vif− virus is pNL4-3 with a deletion in the vif open reading frame (21).

Article Snippet: Size-separated cellular proteins or virion-associated proteins were transferred to polyvinylidene difluoride membranes with a Bio-Rad (Hercules, Calif.) mini-blotting apparatus and probed with a rabbit anti-cyclophilin A antibody (Affinity BioReagents, Heshanic Station, N.J.) or with a murine monoclonal anti-HIV-1 capsid antibody (Dupont NEN).

Techniques: Infection, Transfection, Mutagenesis

Journal: PLoS ONE

Article Title: The Interaction between Circulating Complement Proteins and Cutaneous Microvascular Endothelial Cells in the Development of Childhood Henoch-Schönlein Purpura

doi: 10.1371/journal.pone.0120411

Figure Lengend Snippet: HMVEC-d were pre-incubated with plasma of patients (N = 30) with acute HSP, plasma of healthy controls (N = 30), or culture medium alone for 48 hr, and then the cells were harvested and analyzed for the expression of C3aR (A) and CD88 (B) by flow cytometry. The expression levels were presented as mean fluorescence intensity (MFI).

Article Snippet: Cells were then harvested by trypsin, washed by PBS, and labeled by PE-conjugated mouse IgG anti-human C3aR, IgG anti-human CD88, or IgG isotype control (BD Biosciences) at 4°C for 30 min. Stained cells were assayed by a FACSCalibur using the CellQuest Software (BD Biosciences).

Techniques: Incubation, Expressing, Flow Cytometry, Fluorescence

Journal: Free radical biology & medicine

Article Title: Posttranslational regulation of CYP2J2 by nitric oxide

doi: 10.1016/j.freeradbiomed.2018.04.576

Figure Lengend Snippet: Huh7 cells transduced with pLX304-CYP2J2v5 were treated with 500 µM DPTA for different time periods or with different concentrations of DPTA for 4 hours. Twenty minutes before harvesting cells, 30 µM Luciferin-2J2/4F12, a CYP2J2 substrate was added to each well to measure the activity of CYP2J2. Produced luciferin in collected culture media was detected as luminescence by adding Luciferin detection reagent from Promega. Cell lysates were subjected to SDS-PAGE and the expression levels of CYP2J2 were determined with an antibody to the V5 tag by Western blotting (A). Fluorescent intensity was quantified using Odyssey FC imaging system and the relative content by time (B) and concentration (C) was plotted as the means ± SD of three independent experiments. Activity was plotted from a single experiment.

Article Snippet: The monoclonal CYP2J2 antibody was obtained from Abnova (Taipei, Taiwan, catalog # H00001573-M01).

Techniques: Transduction, Activity Assay, Produced, SDS Page, Expressing, Western Blot, Imaging, Concentration Assay

Journal: Free radical biology & medicine

Article Title: Posttranslational regulation of CYP2J2 by nitric oxide

doi: 10.1016/j.freeradbiomed.2018.04.576

Figure Lengend Snippet: Cells were incubated with 500 µM DPTA for 0, 2, 4 and 6 hours. After the indicated time period, total RNA was extracted and reverse transcribed. The level of CYP2J2 mRNA was determined by RT-PCR and normalized by GAPDH. Values represent the mean ± SD of values from three independent experiments. Differences between 0 h and each time point group were analyzed by one-way ANOVA and Dunnett’s test. No statistical significance was detected.

Article Snippet: The monoclonal CYP2J2 antibody was obtained from Abnova (Taipei, Taiwan, catalog # H00001573-M01).

Techniques: Incubation, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction

Journal: Free radical biology & medicine

Article Title: Posttranslational regulation of CYP2J2 by nitric oxide

doi: 10.1016/j.freeradbiomed.2018.04.576

Figure Lengend Snippet: Huh7 cells were treated with 500 µM DPTA in the presence or absence of 10 µg/ml of the protein synthesis inhibitor cycloheximide (CHX) for indicated time periods. Protein levels of CYP2J2 were analyzed by Western blotting (A) and relative levels of the quantified fluorescent intensity were plotted with the means ± SD of three independent experiments (B). ** p<0.01; ****p <0.0001 versus control (Con) cells at same time point; two-way ANOVA with Sidak’s correction.

Article Snippet: The monoclonal CYP2J2 antibody was obtained from Abnova (Taipei, Taiwan, catalog # H00001573-M01).

Techniques: Western Blot, Control

Journal: Free radical biology & medicine

Article Title: Posttranslational regulation of CYP2J2 by nitric oxide

doi: 10.1016/j.freeradbiomed.2018.04.576

Figure Lengend Snippet: To test the effect of physiological NO on CYP2J2 down-regulation, HeLa cells co-expressing CYP2J2 and doxycycline (Dox) inducible nitric oxide synthase (NOS) were used. Cells were incubated with 10 µM Dox and after 24 h, extracellular media were collected to measure the level of nitrate/nitrite (NOx) before harvesting cells. To inhibit NOS activity, 200 µM L-NAME (LN) was co-incubated with Dox. CYP2J2 levels were measured by Western blotting (A) and the relative expression of CYP2J2 was plotted after normalization using actin (B). NOx levels were determined by Griess reaction (C). ****p < 0.0001 compared to control group; one-way ANOVA and Tukey’s test (n=3).

Article Snippet: The monoclonal CYP2J2 antibody was obtained from Abnova (Taipei, Taiwan, catalog # H00001573-M01).

Techniques: Expressing, Incubation, Activity Assay, Western Blot, Control

Journal: Free radical biology & medicine

Article Title: Posttranslational regulation of CYP2J2 by nitric oxide

doi: 10.1016/j.freeradbiomed.2018.04.576

Figure Lengend Snippet: Two groups of Huh7 cells were treated with 500 µM DPTA for 20, 40, 60, 80 and 100 min. After the indicated time points, in one group 30 µM luciferin-2J2/4F12 was added to cell culture media. In the other group, cells were washed with fresh media twice and then the substrate was added. Cells were incubated with the substrate at 37 °C for 20 min and 25 µl of the reaction mixture was transferred to opaque white 96-well plates. Same volumes of Luciferin detection reagent were added and after 20 minutes, the luminescence was read using the FLUOstar OMEGA microplate reader (A). ****Significantly different from the activity in the washing group at same time point; two-way ANOVA with Sidak’s correction (p < 0.0001; n=3). To determine the contribution of stable NO modifications to CYP2J2 degradation, cells were pre-incubated with DPTA for 4 h in the presence of 10 µg/ml CHX. After washing cells, fresh media without DPTA, and containing 10 µg/ml CHX were added and cells were additionally incubated for 1, 2, 4 or 6 more hours. Measurement of CYP2J2 activity (B) and CYP2J2 protein levels by western blotting (C). Fluorescent intensities from panel C were combined with data from two other experiments and plotted as the means ± SD of three independent experiments (D). # p < 0.05; ## p < 0.01; ### p < 0.001, compared to the 0 h DPTA-treated sample. Two-way ANOVA and Tukey’s test.

Article Snippet: The monoclonal CYP2J2 antibody was obtained from Abnova (Taipei, Taiwan, catalog # H00001573-M01).

Techniques: Cell Culture, Incubation, Activity Assay, Western Blot

Journal: Free radical biology & medicine

Article Title: Posttranslational regulation of CYP2J2 by nitric oxide

doi: 10.1016/j.freeradbiomed.2018.04.576

Figure Lengend Snippet: Huh7 cells were pretreated with 10 µM bortezomib (Bort), a proteasome inhibitor for 1 h, followed by incubation with or without 500 µM DPTA for 4 h. Cell lysates were subjected to western blotting for CYP2J2 expression (A). After normalization of CYP2J2 protein expression using actin and GAPDH, the mean value of each group ± SD was plotted (B). Cellular CYP2J2 catalytic activity (C). To examine ubiquitination of CYP2J2, higher molecular bands were visualized by increasing the gain on the green IR fluorescence signal (D). After 2 h DPTA treatment, ubiquitination of CYP2J2 was confirmed by immunoprecipitation of CYP2J2v5 in HA-Ub transfected cells followed by immunoblotting against HA-Ub (E). **** p < 0.0001, compared to control; # p < 0.05, compared to DPTA. One-way ANOVA and Tukey’s test

Article Snippet: The monoclonal CYP2J2 antibody was obtained from Abnova (Taipei, Taiwan, catalog # H00001573-M01).

Techniques: Incubation, Western Blot, Expressing, Activity Assay, Ubiquitin Proteomics, Fluorescence, Immunoprecipitation, Transfection, Control

Journal: Free radical biology & medicine

Article Title: Posttranslational regulation of CYP2J2 by nitric oxide

doi: 10.1016/j.freeradbiomed.2018.04.576

Figure Lengend Snippet: Huh7 cells were incubated with 500 µM DPTA for 4h following pretreatment of 50 µM calpeptin (Calp), a calpain inhibitor, or 10 µM danazol (Dan), a potent CYP2J2 inhibitor for 1 h. CYP2J2 levels were determined by Western blotting (A, D) and the mean value of each group ± SD from at least three independent experiments was plotted after normalization using GAPDH and actin (B, E). Activity was measured as described in Materials and Methods. ** p < 0.001 and **** p < 0.0001, compared to control; # p < 0.05 and ## p < 0.01, compared to DPTA. One-way ANOVA and Tukey’s test.

Article Snippet: The monoclonal CYP2J2 antibody was obtained from Abnova (Taipei, Taiwan, catalog # H00001573-M01).

Techniques: Incubation, Western Blot, Activity Assay, Control