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Journal: Cell Death & Disease
Article Title: Stabilization of SAMHD1 by NONO is crucial for Ara-C resistance in AML
doi: 10.1038/s41419-022-05023-0
Figure Lengend Snippet: A , B Overexpression or silencing of DCAF1 decreased or increased SAMHD1 expression, respectively. A HL60 cells were transfected with increasing concentrations of HA-tagged DCAF1 24 h, and the levels of the indicated proteins were determined by western blotting. B THP-1 cells were transfected with DCAF1 siRNAs for 24 h, and the levels of the indicated proteins were then determined by western blotting. C DCAF1 interacts with SAMHD1. 293 T cells were transfected with HA-tagged DCAF1 and Myc-tagged SAMHD1 for 24 h. Co-IP was performed with anti-Myc agarose, and the interactions were examined by western blotting. D Overexpression of DCAF1 increased the ubiquitination level of SAMHD1. Myc-tagged SAMHD1 was cotransfected with HA-tagged DCAF1, Flag-ubiquitin or empty vector into 293 T cells for 24 h, and the ubiquitination level of SAMHD1 was determined using IP followed by western blotting. E NONO blocked DCAF1-mediated degradation of SAMHD1. Myc-tagged SAMHD1 and HA-tagged DCAF1 were cotransfected into 293 T cells with or without overexpression of HA-tagged NONO for 24 h. The levels of the indicated proteins were determined by western blotting. F Overexpression of NONO inhibited DCAF1-mediated ubiquitination of SAMHD1. Myc-tagged SAMHD1, HA-tagged DCAF1, and Flag-tagged ubiquitin were cotransfected into 293 T cells with or without overexpression of HA-tagged NONO for 24 h. The ubiquitination of SAMHD1 was evaluated by IP followed by western blotting. G NONO inhibits the interaction of DCAF1 and SAMHD1. Myc-tagged SAMHD1 and HA-tagged DCAF1 were cotransfected into 293 T cells with or without overexpression of Myc-tagged NONO for 24 h. Cells were subsequently subjected to co-IP followed by western blotting with the indicated antibodies. H Silencing of NONO increases the interaction of DCAF1 with SAMHD1. THP-1 cells silencing NONO for 48 h were lysed and were subsequently subjected to co-IP followed by western blotting with the indicated antibodies. For respective immunoblots, the protein levels were quantified by ImageJ software.
Article Snippet: The antibodies against NONO (11058-1-AP), SAMHD1 (12586-1-AP),
Techniques: Over Expression, Expressing, Transfection, Western Blot, Co-Immunoprecipitation Assay, Plasmid Preparation, Software
Journal: Cell Reports
Article Title: HIV-1 Vpr drives a tissue residency-like phenotype during selective infection of resting memory T cells
doi: 10.1016/j.celrep.2022.110650
Figure Lengend Snippet:
Article Snippet:
Techniques: Purification, Isolation, Diagnostic Assay, Recombinant, Derivative Assay, Transfection, Staining, Lysis, SYBR Green Assay, Cell Isolation, Software
Journal: Cell Reports
Article Title: HIV-1 Vpr drives a tissue residency-like phenotype during selective infection of resting memory T cells
doi: 10.1016/j.celrep.2022.110650
Figure Lengend Snippet:
Article Snippet: Blots were probed with rabbit antisera raised against HIV-1 Gag p24 (cat# 0432 donated by Dr G. Reid and obtained from the CFAR), Vpr anti-serum (cat# 3951, NIH ARP), α−P-STAT5 (Tyr694) (D47E7, Cell Signaling Technology), α-STAT5 (D3N2B, Cell Signaling Technology), α-beta-Actin (A2066, Sigma-Aldrich), α-UNG (OTI2C12, OriGene Technologies), α−alpha-Tubulin (T6199, Sigma-Aldrich) and
Techniques: Purification, Isolation, Diagnostic Assay, Recombinant, Derivative Assay, Transfection, Staining, Lysis, SYBR Green Assay, Cell Isolation, Software
Journal: Viruses
Article Title: Huntingtin-Interacting Protein 1 Promotes Vpr-Induced G2 Arrest and HIV-1 Infection in Macrophages
doi: 10.3390/v13112308
Figure Lengend Snippet: Vpr interacts with HIP1. ( a ) GST and GST-Vpr were resolved by 12% SDS-PAGE and stained with Coomassie Brilliant Blue (CBB) (left). Glutathione-Sepharose beads combined with the GST-Vpr or GST alone were incubated with purified HA-HIP1 protein. The bound fractions and 10% of the input were analyzed by Western blot using the anti-HA mAb (right). The positions of HA-HIP1, GST-Vpr, and GST are indicated. ( b ) 293T cells were co-transfected with pME/FVpr and pCAGGS/HA-HIP1 or control pCAGGS/HA. At 48-h post-transfection, cells were lysed, and the lysates were subjected to immunoprecipitation assays using anti-HA agarose and the HA peptide. The bound fractions and inputs were analyzed by Western blotting using the anti-HA polyclonal Ab, anti-Flag polyclonal Ab, and anti-β-actin mAb.
Article Snippet: Equal amounts of total protein were examined by Western blotting using the following antibodies: anti-Flag monoclonal antibody (mAb; M2; Sigma-Aldrich), anti-Flag polyclonal antibody (Sigma-Aldrich), anti-HA polyclonal antibody (Sigma-Aldrich), anti-β-actin mAb (Sigma-Aldrich), anti-HA mAb (MBL International, Woburn, MA, USA), anti-HIP1 mAb (Novus Biologicals, Littleton, CO, USA),
Techniques: SDS Page, Staining, Incubation, Purification, Western Blot, Transfection, Immunoprecipitation
Journal: PLoS ONE
Article Title: The CRL4 VPRBP(DCAF1) E3 ubiquitin ligase directs constitutive RAG1 degradation in a non-lymphoid cell line
doi: 10.1371/journal.pone.0258683
Figure Lengend Snippet: MLN4924 treatment does not block interaction between RAG1 and CRL4 VprBP .
Article Snippet: The following primary antibodies were used:
Techniques: Blocking Assay
Journal: The Journal of Clinical Investigation
Article Title: DCAF1 regulates Treg senescence via the ROS axis during immunological aging
doi: 10.1172/JCI136466
Figure Lengend Snippet: (A) Protein expression of DCAF1 in Tregs isolated from young and aged mice, assessed by immunoblotting. Left: Representative of 3 independent experiments. Right: Statistical summary, means ± SD, *P < 0.05, by Mann-Whitney U test. (B) SA-β-gal activity in CD4+CD25+ Tregs and CD4+CD25– Tconv cells in splenocytes from mice of indicated genotypes, analyzed by flow cytometry with the fluorescent β-gal substrate C12FDG (gray area, no C12FDG; n = 6 mice of 3 experiments; representative results are shown; means ± SD, ****P < 0.0001, by 1-way ANOVA followed by Tukey’s multiple-comparisons test). (C) Heatmap analysis of RNA-Seq data sets to compare top regulated genes in young WT, young Dcaf1-deficient (KO), and aged WT Tregs. The depicted distance was calculated based on Pearson’s correlation. (D) Upregulation of the aging program in Dcaf1-deficient (KO) Tregs (left) and Tconv cells (right), revealed by GSEA of RNA-Seq data sets. (E) Comparison of aging signature gene expression in indicated T cells by qRT-PCR analysis of indicated genes (n = 10 mice of 4 experiments; means ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by 1-way ANOVA followed by Tukey’s multiple-comparisons test).
Article Snippet: The soluble fraction was divided into 2 parts and incubated with magnetic beads that conjugated with
Techniques: Expressing, Isolation, Western Blot, MANN-WHITNEY, Activity Assay, Flow Cytometry, RNA Sequencing Assay, Quantitative RT-PCR
Journal: The Journal of Clinical Investigation
Article Title: DCAF1 regulates Treg senescence via the ROS axis during immunological aging
doi: 10.1172/JCI136466
Figure Lengend Snippet: (A) The protein expression of DCAF1 and β-actin in human 293T cells transduced with lentivirus expressing 2 shRNAs targeting Dcaf1 and scrambled control for 4 days. The results are representative of 3 independent experiments. (B) Flow cytometry of ROS level in human T cells transduced with lentivirus expressing 2 shRNAs targeting Dcaf1 and scrambled control for 4 days, analyzed by 2′,7′-dichlorofluorescin diacetate (DCFDA) (gray area, no DCFDA; n = 4; representative results of 2 independent experiments are shown; means ± SD, **P < 0.01, by 1-way ANOVA followed by Tukey’s multiple-comparisons test). (C) SA-β-gal activity in human T cells transduced with lentivirus expressing 2 shRNAs targeting Dcaf1 and scrambled control for 6 days, assessed by flow cytometry with the fluorescent β-gal substrate C12FDG (gray area, no C12FDG; n = 4; representative flow cytometry results are shown; means ± SD, **P < 0.01, ***P < 0.001, by 1-way ANOVA followed by Tukey’s multiple-comparisons test). (D) qRT-PCR analysis to determine mRNA expression of p16Ink4a in human T cells transduced with 2 shRNAs targeting Dcaf1 and scrambled control for 6 days (n = 6 from 2 independent experiments; means ± SD, **P < 0.01, ****P < 0.0001, by 1-way ANOVA followed by Tukey’s multiple-comparisons test).
Article Snippet: The soluble fraction was divided into 2 parts and incubated with magnetic beads that conjugated with
Techniques: Expressing, Transduction, Flow Cytometry, Activity Assay, Quantitative RT-PCR
Journal: The Journal of Clinical Investigation
Article Title: DCAF1 regulates Treg senescence via the ROS axis during immunological aging
doi: 10.1172/JCI136466
Figure Lengend Snippet: (A) Pathways commonly enriched in aged and Dcaf1-deficient (CD4-Cre Dcaf1fl/fl) Tregs based on GSEA of RNA-Seq data sets (FDR < 0.05). (B) Enrichment of ROS pathway in aged versus young WT Tregs (top) and Dcaf1-deficient (KO) versus WT Tregs (bottom) by GSEA of RNA-Seq data sets. (C–E) Flow cytometry of ROS level in indicated T cell populations from young WT and aged WT mice (C), young Dcaf1-deficient mice (D) and activated WT and ER-Cre Dcaf1fl/fl CD4+ T cells treated with 4-hydroxy-tamoxifen for indicated days (E), analyzed by DCFDA (gray area, no DCFDA; n = 3 mice of 3 experiments; representative results are shown; means ± SD, **P < 0.01, ***P < 0.001, ****P < 0.0001, by 2-way ANOVA followed by Holm-Šidák multiple-comparisons test). (F and G) Interaction of GSTP1 and DCAF1 by coimmunoprecipitation in 293T cells (F) and by endogenous immunoprecipitation using anti-DCAF1 antibody in mouse T cells (G). The results are representative of 3 independent experiments. (H) GST activity in 293T cells after overexpression of GSTP1 and DCAF1 for 4 days; n = 5; means ± SD, *P < 0.05, **P < 0.01, by Mann-Whitney U test. (I) Flow cytometry of ROS level in activated WT and ER-Cre Dcaf1fl/fl CD4+ T cells transduced with MIT (MSCV-IRES-Thy1.1) or MIT-GSTP1 virus in the presence of 4-hydroxy-tamoxifen, analyzed by DCFDA (gray area, no DCFDA; n = 3–4 experiments; means ± SD, *P < 0.01, **P < 0.05, by 2-way ANOVA followed by Holm-Šidák multiple-comparisons test). (J) Proliferation assayed by BrdU incorporation in young and aged Tregs transduced with MIT or MIT-Gstp1 virus (n = 3 experiments; means ± SD, **P < 0.01, by 1-way ANOVA followed by Tukey’s multiple-comparisons test).
Article Snippet: The soluble fraction was divided into 2 parts and incubated with magnetic beads that conjugated with
Techniques: RNA Sequencing Assay, Flow Cytometry, Immunoprecipitation, Activity Assay, Over Expression, MANN-WHITNEY, Transduction, BrdU Incorporation Assay
Journal: The Journal of Clinical Investigation
Article Title: DCAF1 regulates Treg senescence via the ROS axis during immunological aging
doi: 10.1172/JCI136466
Figure Lengend Snippet: (A and B) Flow cytometry of ROS levels in aged Tregs (A) and Dcaf1-deficient (CD4-Cre Dcaf1fl/fl) Tregs (B) in the absence (–) or presence (+) of NAC (20 mM) or GSH (10 mM) (blank, no DCFDA; n = 3 mice of 3 experiments; representative results are shown; means ± SD, ****P < 0.0001, by 1-way ANOVA followed by Tukey’s multiple-comparisons test). (C) Proliferation assayed by BrdU incorporation in aged Tregs in the absence (–) or presence (+) of NAC (20 mM) or GSH (10 mM) (n = 4 mice of 4 experiments; representative results are shown; means ± SD, ****P < 0.0001, by 1-way ANOVA followed by Tukey’s multiple-comparisons test). (D) Proliferation assayed by BrdU incorporation in Dcaf1-deficient (CD4-Cre Dcaf1fl/fl) Tregs in the absence (–) or presence (+) of NAC (20 mM) or GSH (10 mM) (n = 4 mice of 4 experiments; representative results are shown; means ± SD, ****P < 0.0001, by 1-way ANOVA followed by Tukey’s multiple-comparisons test). (E) Suppressive activity of aged Tregs without (–) or with (+) pretreatment of NAC (20 mM) or GSH (10 mM), assessed by in vitro suppression assay (n = 3 mice of 3 experiments; representative results are shown; means ± SD, ****P < 0.0001, by 1-way ANOVA followed by Tukey’s multiple-comparisons test). (F) Suppressive activity of Dcaf1-deficient (CD4-Cre Dcaf1fl/fl) Tregs without (–) or with (+) pretreatment of NAC (20 mM) or GSH (10 mM), assessed by in vitro suppression assays (n = 3 mice of 3 experiments; representative results are shown; means ± SD, ***P < 0.001, by 1-way ANOVA followed by Tukey’s multiple-comparisons test).
Article Snippet: The soluble fraction was divided into 2 parts and incubated with magnetic beads that conjugated with
Techniques: Flow Cytometry, BrdU Incorporation Assay, Activity Assay, In Vitro, Suppression Assay