goat polyclonal anti-ddb1 antibodies  (Absolute Biotech Inc)

Journal: Journal of Virology

Article Title: Smc5/6 Antagonism by HBx Is an Evolutionarily Conserved Function of Hepatitis B Virus Infection in Mammals

doi: 10.1128/JVI.00769-18

Figure Lengend Snippet: Evolutionary analyses of divergent mammalian HBV X proteins. (A) Phylogenetic analysis of the X proteins from hepadnaviruses that naturally infect mammals. The viral X proteins tested in our in vitro functional assays (Fig. 5 to ​to7)7) are indicated by an asterisk. Phylogenetic analysis of orthohepadnaviral X proteins was performed using a 161-amino-acid alignment obtained with MUSCLE (see supplemental data set 2 at https://figshare.com/articles/DatasetS2_Orthohepadnaviral_HBx_amino_acid_alignment_interleaved_phylip_format_/6194825) and the tree was built with PhyML and a JTT+I+G model with 1,000 bootstrap replicates. Bootstrap values (>800/1,000) are indicated at the nodes. The tree was rooted for representation purposes according to the work of Drexler et al. (52) (but the outgroup of orthohepadnavirus is still under debate [2]). The scale bar indicates the number of amino acid substitutions per site. We analyzed the X proteins from HBVs from the ground squirrel (GSHBV), arctic squirrel (ASHBV), and woodchuck (WHV), three bat viruses naturally infecting Hipposideros cf. ruber (roundleaf bat), Rhinolophus alcyone (horseshoe bat), and Uroderma bilobatum (tent-making bat), respectively (RBHBV, HBHBV, and TBHBV), wooly monkey HBV (WMHBV), human HBV, and HBVs from other indicated hominoids. (B) Amino acid alignment of the viral X proteins used for Fig. 5 to ​to7.7. The black-to-white gradient depicts high-to-low sequence identity (Geneious). The open reading frames (ORFs) overlapping with HBx are shown, as well as the DDB1-binding region in the human viral HBx protein (72).

Article Snippet: The membranes were probed with 1:5,000 mouse monoclonal anti-GFP antibody (Roche; 11814460001) to detect the GFP-tagged X proteins, 1:1,000 mouse monoclonal antibodies against Smc6 (Abgent; AT3956a), 1:500 rabbit polyclonal antibodies against Smc6 (a gift from A. R. Lehmann) (NIH 3T3 [ ]) ( 64 ), 1:1,000 rabbit polyclonal anti-Nse4 (Abgent; AP9909A), 1:10,000 mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; Sigma-Aldrich; G8795), and 1:500 goat polyclonal anti-DDB1 (Everest Biotech) antibodies.

Techniques: In Vitro, Functional Assay, Sequencing, Binding Assay

Journal: Journal of Virology

Article Title: Smc5/6 Antagonism by HBx Is an Evolutionarily Conserved Function of Hepatitis B Virus Infection in Mammals

doi: 10.1128/JVI.00769-18

Figure Lengend Snippet: Highly divergent mammalian HBV X proteins show a conserved property of recruiting human DDB1 and antagonizing human Smc5/6 restriction. (A and B) Degradation of the human Smc5/6 complex by mammalian hepadnavirus X proteins. Human hepatoma HepG2 cells (A) and 293T cells (B) were transduced with a lentivector expressing only GFP (control) or the GFP-fused X protein from diverse hepadnaviruses (Fig. 4) or a mock control. Western blot analysis of the endogenous Smc6 and Nsmce4A was performed (see Materials and Methods). GAPDH served as a loading control. (C) Effect of mammalian X proteins on transiently transfected reporter gene activity. HepG2 cells were transfected with a luciferase reporter construct and the next day transduced with lentiviral vectors expressing the indicated proteins as described above. At days 5 to 7, the luciferase activity was measured; the fold increase of relative light units (RLU) versus the GFP control condition (set at 1) is shown. The means from three independent experiments are shown, along with SDs. *, P value = 0.1. P values correspond to the Wilcoxon Mann-Whitney test against the null hypothesis of no difference in the luciferase activity between the GFP control and GFP-X conditions. Of note, the same six X proteins unfused to GFP (i.e., in their native forms) also retained this activity (data not shown). (D) Interaction with human DDB1 protein was conserved for all hepadnaviral X proteins tested. The presence of DDB1 and GFP-fused protein (IP) was assessed by Western blotting. The viral X proteins could all interact with human DDB1, except for the DDB1 binding-deficient HBx mutant (R96E) that was used as a control. Note that GFP migrates to a position near the immunoglobulin light chain.

Article Snippet: The membranes were probed with 1:5,000 mouse monoclonal anti-GFP antibody (Roche; 11814460001) to detect the GFP-tagged X proteins, 1:1,000 mouse monoclonal antibodies against Smc6 (Abgent; AT3956a), 1:500 rabbit polyclonal antibodies against Smc6 (a gift from A. R. Lehmann) (NIH 3T3 [ ]) ( 64 ), 1:1,000 rabbit polyclonal anti-Nse4 (Abgent; AP9909A), 1:10,000 mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH; Sigma-Aldrich; G8795), and 1:500 goat polyclonal anti-DDB1 (Everest Biotech) antibodies.

Techniques: Transduction, Expressing, Western Blot, Transfection, Activity Assay, Luciferase, Construct, MANN-WHITNEY, Binding Assay, Mutagenesis

Journal: Molecular and Cellular Biology

Article Title: Cellular Concentrations of DDB2 Regulate Dynamic Binding of DDB1 at UV-Induced DNA Damage

doi: 10.1128/mcb.01108-08

Figure Lengend Snippet: FIG. 1. Expression and subcellular localization of mCherry-tagged DDB1. (A) Immunoblot analysis of parental VH10SV40 cells and VH10SV40 cells stably expressing mCherry-DDB1 (A634), using antibodies against DDB1. (B) Subcellular localization of mCherry-DDB1 in A634 cells. (C) Subcellular localization of endogenous DDB1 in cells detected by immunostaining of VH10SV40 cells by use of antibodies against DDB1. (D) Subcellular localization of mCherry-DDB1 in VH10SV40 cells transiently transfected with the mCherry-DDB1-expressing construct (48 h after transfection). (E) Nuclear localization of mCherry-DDB1 in VH10SV40 cells transiently cotransfected with mCherry-DDB1- and eYFP-DDB2- expressing constructs (48 h after transfection).

Article Snippet: DDB1 was detected with goat polyclonal anti-DDB1 antibodies (1:1,000; Abcam), and CUL4A was detected with rabbit polyclonal CUL4A antibodies (1:1,000; Abcam).

Techniques: Expressing, Western Blot, Stable Transfection, Immunostaining, Transfection, Construct

Journal: Molecular and Cellular Biology

Article Title: Cellular Concentrations of DDB2 Regulate Dynamic Binding of DDB1 at UV-Induced DNA Damage

doi: 10.1128/mcb.01108-08

Figure Lengend Snippet: FIG. 2. Recruitment of mCherry-DDB1 to UV-damaged DNA. (A) Accumulation of mCherry-DDB1 at the site of local damage in A634 cells (indicated with an arrow) irradiated with 100 J/m2 of UV-C through a polycarbonate filter (5 min after UV irradiation). (B) Quan- tification of the accumulation kinetics of mCherry-DDB1, eYFP- DDB2, and GFP-CUL4A at the site of local damage. Curves were normalized to the plateau value. Time point 0 corresponds to the beginning of the UV irradiation. (C) FRAP analysis of mCherry- DDB1 mobility in mock-irradiated (M.I.) and UV-irradiated (10 J/m2) A634 cells. Experimental curves, curves obtained by Monte Carlo simulations (sim.), and residuals (res.) are shown.

Article Snippet: DDB1 was detected with goat polyclonal anti-DDB1 antibodies (1:1,000; Abcam), and CUL4A was detected with rabbit polyclonal CUL4A antibodies (1:1,000; Abcam).

Techniques: Irradiation

Journal: Molecular and Cellular Biology

Article Title: Cellular Concentrations of DDB2 Regulate Dynamic Binding of DDB1 at UV-Induced DNA Damage

doi: 10.1128/mcb.01108-08

Figure Lengend Snippet: FIG. 3. Factors influencing UV-induced immobilization of DDB1. (A) Immobilization of mCherry-DDB1 in A634 cells after mock irradiation (M.I.) or different doses of UV. (B) Immobilization of mCherry-DDB1 in A634 cells transiently overexpressing eYFP-DDB2 after different doses of UV-C. (C) Electrophoretic mobility shift assay showing reduction of damaged DNA binding activity in A634 cells transfected with siRNA against DDB2 (DDB2 siRNA) or control nontargeting siRNA (Ct siRNA). (D) Immobile fraction of mCherry-DDB1 in A634 cells transfected with siRNA against DDB2 or control nontargeting siRNA after mock irradiation or irradiation with 10 J/m2 of UV-C. (E) Western blot analysis of CUL4A depletion by siRNA against CUL4A. (F) Immobile fraction of mCherry-DDB1 in A634 cells transfected with siRNA against CUL4A or control (nontargeting) siRNA (mock irradiated or irradiated with 10 J/m2 of UV-C).

Article Snippet: DDB1 was detected with goat polyclonal anti-DDB1 antibodies (1:1,000; Abcam), and CUL4A was detected with rabbit polyclonal CUL4A antibodies (1:1,000; Abcam).

Techniques: Irradiation, Electrophoretic Mobility Shift Assay, Binding Assay, Activity Assay, Transfection, Control, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Cellular Concentrations of DDB2 Regulate Dynamic Binding of DDB1 at UV-Induced DNA Damage

doi: 10.1128/mcb.01108-08

Figure Lengend Snippet: FIG. 4. Decrease of UV-dependent DDB1 immobilization with time. (A) UV-dependent mCherry-DDB1 immobilization after 10, 20, and 40 J/m2 of UV-C at different time points after UV irradiation or mock irradiation (M.I.). (B) UV-dependent mCherry-DDB1 immobi- lization in XP-C and XP-A cells after 10 J/m2 of UV-C at different time points after UV irradiation or mock irradiation. (C) UV-dependent mCherry-DDB1 immobilization at different time points after 10 J/m2

Article Snippet: DDB1 was detected with goat polyclonal anti-DDB1 antibodies (1:1,000; Abcam), and CUL4A was detected with rabbit polyclonal CUL4A antibodies (1:1,000; Abcam).

Techniques: Irradiation

Journal: Molecular and Cellular Biology

Article Title: Cellular Concentrations of DDB2 Regulate Dynamic Binding of DDB1 at UV-Induced DNA Damage

doi: 10.1128/mcb.01108-08

Figure Lengend Snippet: FIG. 5. Residence time and dissociation kinetics of DDB1 and DDB2 proteins on local DNA damage. (A) Illustration of FRAP-LD in wild-type cells expressing mCherry-DDB1 and eYFP-DDB2. A cell is shown before the bleaching and at different time points after the bleaching. (B) FRAP-LD analysis of mCherry-DDB1 on local DNA damage in the nuclei of wild-type (WT) cells, XP-C cells, and wild-type cells treated with the proteasome inhibitor MG-132. (C) FRAP-LD analysis of eYFP-DDB2 on local DNA damage in the nuclei of wild-type cells, XP-C cells, and wild-type cells treated with the proteasome inhibitor MG-132. (D) Illustration of FLIP in wild-type cells expressing mCherry-DDB1 and eYFP-DDB2. A cell is shown before the bleaching and at different time points after the beginning of the repeated bleachings. (E) Dissociation kinetics of mCherry-DDB1 on local DNA damage in the nuclei of wild-type cells, XP-C cells, and wild-type cells treated with the proteasome inhibitor MG-132, as measured by FLIP. Also shown is a FLIP curve for XPC-eGFP. Error bars represent standard errors of the means. FRAP-LD and FLIP curves are based on 9 to 12 cells.

Article Snippet: DDB1 was detected with goat polyclonal anti-DDB1 antibodies (1:1,000; Abcam), and CUL4A was detected with rabbit polyclonal CUL4A antibodies (1:1,000; Abcam).

Techniques: Expressing

Journal: Molecular and Cellular Biology

Article Title: Cellular Concentrations of DDB2 Regulate Dynamic Binding of DDB1 at UV-Induced DNA Damage

doi: 10.1128/mcb.01108-08

Figure Lengend Snippet: FIG. 6. Hypothesis proposing the role of UV-induced DDB2 degradation in release of DDB1 for the interactions outside NER. Without UV irradiation, different WD40 repeat proteins associate with DDB1-ubiquitine ligase complexes. In the presence of the unrepaired DNA lesion, equilibrium is shifted toward assembly of the DDB2-DDB1-CUL4A-ROC1 complex, which constantly rebinds to the damaged DNA. At the same time, binding of the DDB2-containing ubiquitin ligase complex to UV-damaged DNA triggers ubiquitylation and proteasomic degradation of DDB2, which makes DDB1 available for inclusion in other E3 complexes, providing cellular response to the genotoxic stress.

Article Snippet: DDB1 was detected with goat polyclonal anti-DDB1 antibodies (1:1,000; Abcam), and CUL4A was detected with rabbit polyclonal CUL4A antibodies (1:1,000; Abcam).

Techniques: Irradiation, Binding Assay, Ubiquitin Proteomics