Journal: iScience

Article Title: The protease DDI2 regulates NRF1 activation in response to cadmium toxicity

doi: 10.1016/j.isci.2022.105227

Figure Lengend Snippet: Cadmium indirectly inhibits the proteasome (A) Relative proteasome activity of HepG2 cells treated with CdCl 2 (Cd; + 1 μM or ++ 5 μM) or carfilzomib (CFZ, 10 nM) during 6 h. Each bar represents the mean, and error bars denote SD of at least three independent experiments. (B) Relative proteasome activity of purified 26S proteasome complexes incubated with the substrate Suc-LLVY-AMC and treated with or without Cd (1 μM) or CFZ (10 nM) during the indicated time in minutes (n = 3).

Article Snippet: Purified human proteasome complexes , Bio-Techne , E−365-025.

Techniques: Activity Assay, Purification, Incubation

Journal: iScience

Article Title: The protease DDI2 regulates NRF1 activation in response to cadmium toxicity

doi: 10.1016/j.isci.2022.105227

Figure Lengend Snippet:

Article Snippet: Purified human proteasome complexes , Bio-Techne , E−365-025.

Techniques: Recombinant, Purification, Bicinchoninic Acid Protein Assay, Activity Assay, MTS Assay, Expressing, Western Blot, Plasmid Preparation, Software

Journal: Human molecular genetics

Article Title: USP1 deubiquitinase maintains phosphorylated CHK1 by limiting its DDB1-dependent degradation.

doi: 10.1093/hmg/ddr103

Figure Lengend Snippet: Figure 5. Phosphorylated CHK1 is degraded in a DDB1-dependent manner in the absence of USP1. (A) U2OS cells were transfected with the indicated siRNAs, treated 72 h later with UVC (10 J/m2) and lysed 4 h later. Proteins were analysed by western blot with the indicated antibodies. (∗) indicates a nonspecific band recognized by the anti-p345 CHK1 antibody. CLS and U1 indicate CLASPIN and USP1, respectively. (B) HeLa cells transfected with the indicated siRNAs were treated with MMC (2 mg/ml/1 h) or UVC (10 J/m2) and lysed 20 h and 4 h later, respectively. The lysates were analysed by western blot with the indicated antibodies. U1 and D1 indicate USP1 and DDB1, respectively. (C) U2OS cells transfected with Ctrl or USP1 siRNAs were treated with DMSO or the proteasome inhibitor MG132 (10 mM) for 6 h. The cellular extracts were analysed by western blot using the indicated antibodies. (D) Endogenous CHK1 was immunopre- cipitated from HEK293T cells that were untreated or treated with HU (5 mM; 4 h), and its association with DDB1 was analysed by western blot. (E) The lysates from HeLa cells transfected with the indicated siRNAs were analysed by immunoblotting to monitor CDT1 levels. DDB1 inhibition stabilized CDT1 while USP1 depletion had no effect. (F) HeLa cells transfected with Ctrl or USP1 siRNAs were treated with MMC (2 mg/ml/1 h) and lysed 20 h later. While USP1 depletion significantly reduced p-CHK1 and protein levels, it did not enhance the degradation of CDT1. (G) A model illustrating the major molecular mechanisms involved in CHK1 regulation and USP1 participation in this phenomenon. The FANCcore complex was voluntarily excluded from the schema to simplify the figure.

Article Snippet: The antibodies used were directed against FANCD2, RAD17, CHK1, actin, NBS1 and PCNA (Santa Cruz Biotechnology); FANCD2, RPA32, SMC1, p966SMC1 and vinculin (Abcam); p645RAD17, p345CHK1 and p343NBS1 (Cell Signalling); p68CHK2 and RPA32 (Calbiochem); CLASPIN, FANCI, DDB1 and CDT1 (Bethyl); and gH2AX (Upstate).

Techniques: Transfection, Western Blot, Inhibition

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 1. DDB1 interacts with and cleaved by NS3/4A. (A) DDB1 interacts with NS3/4A in overexpression system. The 293 cells were transfected with the indicated plasmids. Coimmunoprecipitation was performed with anti-Flag or control IgG. The immunoprecipitates were analyzed by immunoblot with anti-Flag anti-HA. The lysates were analyzed by immunoblots with anti-DDB1 or anti-HA. (B) Endogenous DDB1 interacts with NS3/4A in JFH-1 infected cells. Huh-7 cells (5 107) were mock-infected or infected with JFH-1 (Multiplicity of Infection, MOI: 0.3) for 3 days. Coimmunoprecipitation was performed with anti-DDB1 or control IgG. The immunoprecipitates were analyzed by immunoblot with anti-DDB1 and anti-NS3. The lysates were analyzed by immunoblots with anti-DDB1 or anti-NS3.

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Over Expression, Transfection, Control, Western Blot, Infection

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 2. NS3/4A cleaves DDB1 at C378. (A) Cleavage of DDB1 by NS3/4A is inhibited by the NS3/4A inhibitor VX-950. The 293 cells were transfected with N-terminal or C-terminal Flag-tagged DDB1 (N-Flag-DDB1 or DDB1-C-Flag respectively) and HA-NS3/4A. The transfected cells were treated with VX-950 (0.2 mM) or left untreated for 1 day before immunoblot analysis with anti-Flag or anti-HA. (B) Alignment of the junction sequences of NS proteins of HCV and the potential NS3/4A cleavage sites in TC-PTP, VISA, TRIF and DDB1. (C) NS3/4A cleaves DDB1 at C378. The 293 cells were transfected with the indicated plasmids and cells lysates were analyzed by immunoblots with anti-Flag or anti-HA. (D) DDB1 N-terminal cleavage product migrated similarly to overexpressed DDB1(1–378) mutant. The 293 cells were transfected with the indicated plasmids, treated with VX-950 or left untreated for 1 day before immunoblot analysis with with anti-Flag or anti-HA.

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Transfection, Western Blot, Mutagenesis

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 3. DDB1 plays a critical role in HCV replication. (A) Overexpression of DDB1 potentiates HCV RNA replication. Huh-7 cells (1 106) were transfected with the indicated amounts of Flag-DDB1 plasmid for 24 h and then cells were split and mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were determined by RT-qPCR and normalized to cellular GAPDH mRNA levels. The uninfected cell lysates were analyzed by immunoblots with anti-Flag or anti-b-actin. Graphs show mean7SD, n¼3. (B) Knockdown of DDB1 inhibits HCV RNA replication. Control or DDB1-RNAi knockdown Huh-7 cells were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were then determined by RT-qPCR and normalized to GAPDH mRNA levels. The uninfected cells lysates were also analyzed by immunoblots with anti-DDB1 or anti-b-actin. Graphs show meanþSD, n¼3. (C) Knockdown of DDB1 inhibits HCV protein expression. Control or DDB1-RNAi knockdown Huh-7 cells were mock-infected or infected with JFH-1 for 3 days, and the cells were then analyzed by immunofluorescent staining with anti-E2 (red), and Hoechst (blue). (D) Knockdown of DDB1 inhibits production of infectious HCV particles. Control or DDB1-RNAi knockdown Huh-7 cells were mock-infected or infected with JFH-1 for 24 h. The cells were completely washed and fresh complete medium was added for 48 h. The JFH-1 infected medium was collected and diluted for infection of Huh-7.5.1 cells. Three days later, cells were analyzed by immunofluorescent staining with anti-E2 and HCV titers were calculated by counting positive stained cells foci. Graphs show mean7SD, n¼3. (E) Knockdown of DDB1 inhibits RNA replication of HCV subgenomic replicon. Control or DDB1-RNAi knockdown Huh-7 cells and Huh-7 Con1 subgenomic replicon cells were cultured for 3 days. The cells (2 106) were collected and intracellular HCV RNA levels were determined by RT-qPCR and normalized to cellular GAPDH mRNA levels. Cell lysates were analyzed by immunoblots with anti-DDB1 or anti-b-actin. Graphs show mean7SD, n¼3. (F) DDB1 has no effects on HCV entry. Control or DDB1-RNAi knockdown Huh-7 cells were infected with HCVpp for 3 days (NC: Negative Control, HCVpp pakaging without HCV E1E2). The lysates of infected cells were assayed by luciferase reporter assays and immunoblots with anti-DDB1 or anti-b-actin. Graphs show mean7SD, n¼3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Over Expression, Transfection, Plasmid Preparation, Infection, Quantitative RT-PCR, Western Blot, Knockdown, Control, Expressing, Staining, Cell Culture, Negative Control, Luciferase

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 4. DDB1 cleavage is required for HCV replication. (A) The indicated stable cell lines were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were then determined by RT-qPCR and normalized to GAPDH mRNA levels. The uninfected cells lysates were also analyzed by immunoblots with anti-DDB1 or anti-b-actin. Graphs show meanþSD, n¼3. (B) The indicated stable cells were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. The cells were then analyzed by immunofluorescent staining with anti-E2 (red), and Hoechst (blue). (C) The indicated stable cell lines were mock-infected or infected with JFH-1 (MOI: 0.3) for 24 h. The cells were completely washed and fresh medium was added for 48 h. The JFH-1-containing medium was collected and diluted for infection of Huh-7.5.1 cells. Three days later, cells were analyzed by immunofluorescent staining with anti-E2 and HCV titers were calculated by counting positive stained cells foci. Graphs show meanþSD, n¼3. (D) Control or DDB1-RNAi knockdown Huh-7 cells were stably transduced with empty vector, DDB1, DDB1(C378R), off-target nonsense mutants of DDB1 or DDB1(C378R) respectively. Two days later, cells were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were then determined by RT-qPCR and normalized to GAPDH mRNA levels. The cells lysates were also analyzed by immunoblots with anti-DDB1 or anti-actin. Graphs show meanþSD, n¼3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Stable Transfection, Infection, Quantitative RT-PCR, Western Blot, Staining, Control, Knockdown, Transduction, Plasmid Preparation

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 5. DDB1 cleavage products do not affect the HCV infection. (A) Huh-7 cells stably transduced with the indicated DDB1 truncation mutants were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were then determined by RT-qPCR and normalized to GAPDH mRNA levels. The uninfected cell lysates were analyzed by immunoblots with anti-DDB1 or anti-b-actin. Graphs show meanþSD, n¼3. (B) Huh-7 cells stably transduced with the indicated DDB1 truncation mutants were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. The cells were then analyzed by immunofluorescent staining with anti-E2 (red), and Hoechst (blue). (C) Huh-7 cells stably transduced with the indicated DDB1 truncation mutants were mock-infected or infected with JFH-1 (MOI: 0.3) for 24 h. The cells were completely washed and fresh complete medium was added for 48 h. The JFH-1 infected medium was collected and diluted for infection of Huh-7.5.1 cells. Three days later, cells were analyzed by immunofluorescent staining with anti-E2 and HCV titers were calculated by counting positive stained cells foci. Graphs show meanþSD, n¼3. (D–F) Control or DDB1-RNAi-#1 (targeted sequence is within the cDNA fragment encoding aa379–1140) transduced Huh-7 cells were further transfected with empty vector, Flag-DDB1(1–378), Flag-DDB1(379–1140*) (*, a RNAi off-target mutant),or a combination of Flag-DDB1(1–378) and Flag-DDB1(379–1140*) by Lipofectamine 2000. One day post transfection, the cells were split and mock infected or infected with JFH-1(MOI: 0.3) for 3 (D, E) or 1 (F) day. Intracellular HCV RNA levels (D), intracellular viral particles (E), or viral titers in the medium (F) were then determined as described in (A–C). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Infection, Stable Transfection, Transduction, Quantitative RT-PCR, Western Blot, Staining, Control, Sequencing, Transfection, Plasmid Preparation, Mutagenesis

Journal: Scientific Reports

Article Title: The Cullin 4A/B-DDB1-Cereblon E3 Ubiquitin Ligase Complex Mediates the Degradation of CLC-1 Chloride Channels

doi: 10.1038/srep10667

Figure Lengend Snippet: Biochemical inspection of CLC-1 protein interactions. (A) Co-immunoprecipitation of HA-CLC-1 and Myc-CUL4A/B in HEK293T cells. Co-expression of HA-CLC-1 and the Myc vector was used as the control experiment. (B) Co-immunoprecipitation of Myc - CLC-1 and Flag-DDB1 in HEK293T cells. Co-expression of Flag-DDB1 and the Myc vector was used as the control experiment. (C) Lack of co-immunoprecipitation of Myc-CLC-1 and Flag-DDB2 in HEK293T cells. Co-expression of Flag-DDB2 and the Myc vector was used as the control experiment. (D) Co-immunoprecipitation of Myc - CLC-1 and HA-CRBN in HEK293T cells. Co-expression of Myc-CLC-1 and the HA vector was used as the control experiment. Cell lysates were immunoprecipitated with the anti-Myc ( A,B,C ) or anti-HA ( D ) antibody. (E) Protein interactions of endogenous CLC-1 channels in rat skeletal muscle. Verification of the specificity of the anti-CLC-1 antibody: ( Far left ) CLC-1 immunoreactivity was notably reduced in the presence of an excess of a control antigen peptide; ( Center left ) immunoprecipitation was achieved by using the anti-CLC-1 antibody, but not rabbit IgG. ( Center right , Far right ) Co-immunoprecipitation of CLC-1, CRBN, DDB1, and CUL4B. Muscle lysates were immunoprecipitated with the anti-CLC-1 antibody. h.c.: IgG heavy chain. The gels were run under the same experimental conditions. Uncropped images of immunoblots are shown in .

Article Snippet: Other cDNA constructs employed in this study include pcDNA3.1-Flag dominant-negative human cullin 1/2/3/4A/4B/5 (Addgene 15818-15823), pcDNA3-Myc human cullin 3/4A/4B (Addgene 19893, 19951, 19922), pcDNA3-HA wild-type and lysine-less human ubiquitin (kindly provided by Dr. Chihiro Sasakawa, University of Tokyo, Japan), pcDNA3-Flag human DDB1 (Addgene 19918), pcDNA3-Flag human DDB2 (kindly provided by Dr. Show-Li Chen, National Taiwan University, Taiwan), and pcDNA3-HA rat cereblon (kindly provided by Dr. Chul-Seung Park, Gwangju Institute of Science and Technology, Korea).

Techniques: Immunoprecipitation, Expressing, Plasmid Preparation, Control, Western Blot

Journal: Scientific Reports

Article Title: The Cullin 4A/B-DDB1-Cereblon E3 Ubiquitin Ligase Complex Mediates the Degradation of CLC-1 Chloride Channels

doi: 10.1038/srep10667

Figure Lengend Snippet: Biochemical demonstration of the regulation of CLC-1 by CRBN in HEK293T cells. (A) (Top) Representative immunoblots showing the effect of Flag-DDB1 or HA-CRBN over-expression on Myc-CLC-1 protein. Co-expression with the Flag/HA vector was used as the control experiment. (Bottom) Quantification of relative CLC-1 protein expression level. Values from the DDB1/CRBN co-expression group ( hatched bars ) were normalized to those for the corresponding vector control ( clear bars ). Asterisks denote significant difference from the control (*, t -test: p < 0.05; n =6-12). (B) CLC-1 polyubiquitination [CLC-1-(Ub)n] by endogenous ubiquitin was enhanced by CRBN over-expression. Cell lysates were immunoprecipitated (IP) with the anti-Myc antibody. Protein ubiquitination was identified by immunoblotting the immunoprecipitates with the anti-ubiquitin (Ub) antibody. (C) Representative immunoblots showing the effect of shRNA knock-down of endogenous DDB1 or CRBN. The numbers denote the relative CLC-1/DDB1/CRBN expression level with respect to the control shRNA for GFP. The gels were run under the same experimental conditions. Uncropped images of immunoblots are shown in .

Article Snippet: Other cDNA constructs employed in this study include pcDNA3.1-Flag dominant-negative human cullin 1/2/3/4A/4B/5 (Addgene 15818-15823), pcDNA3-Myc human cullin 3/4A/4B (Addgene 19893, 19951, 19922), pcDNA3-HA wild-type and lysine-less human ubiquitin (kindly provided by Dr. Chihiro Sasakawa, University of Tokyo, Japan), pcDNA3-Flag human DDB1 (Addgene 19918), pcDNA3-Flag human DDB2 (kindly provided by Dr. Show-Li Chen, National Taiwan University, Taiwan), and pcDNA3-HA rat cereblon (kindly provided by Dr. Chul-Seung Park, Gwangju Institute of Science and Technology, Korea).

Techniques: Western Blot, Over Expression, Expressing, Plasmid Preparation, Control, Ubiquitin Proteomics, Immunoprecipitation, shRNA, Knockdown

Journal: Genes & Development

Article Title: A cryptic Tudor domain links BRWD2/PHIP to COMPASS-mediated histone H3K4 methylation

doi: 10.1101/gad.305201.117

Figure Lengend Snippet: BRWD2/PHIP associates with CRL4, chromatin regulators, and active chromatin marks. ( A ) Silver-stained SDS-PAGE gel of Flag immunoprecipitations from control parental HEK293 TRex-expressing and Flag-BRWD2/PHIP-expressing cells. ( B ) Diagram summarizing BRWD2/PHIP protein–protein interactions. The solid black line indicates a strong interaction between BRWD2/PHIP and DDB1–CUL4A/B. Black dashed lines indicate interactions with chromatin remodelers and histones. Red dashed lines indicate interactions sensitive to treatment with MLN4924. ( C ) Western blot confirming BRWD2/PHIP interaction with CRL4 (DDB1 and CUL4A) and chromatin remodelers (CHD4 and PHF6). The middle band detected in the BRWD2/PHIP Western likely represents a BRWD2/PHIP protein cleavage product that occurs during immunoprecipitation incubation. ( D ) Western blot detecting histone modifications (H3K27ac, H3K4me1, H3K4me3, and H3K27me3) and histone variants (H2A.Z) associated with BRWD2/PHIP. Note that Flag-BRWD2/PHIP is expressed at near-endogenous levels and is not detectable with anti-Flag in the input but is detected after enrichment by immunoprecipitation.

Article Snippet: Other antibodies used in this study were H3K4me1 (Shilatifard Laboratory, no. 24), H3K4me2 (Shilatifard Laboratory, no. 27), H3K4me3 (Shilatifard Laboratory, no. 680), H3K27ac (Cell Signaling Technology, no. 8137), H2A.Z (Cell Signaling Technology, no. 2718), H3K27me3 (Shilatifard Laboratory, no. 67), DDB1 (Cell Signaling Technology, no. 6998), CUL4A (Cell Signaling Technology, no. 2699), CHD4 (Cell Signaling Technology #12011), PHF6 (Santa Cruz Biotechnology, no. sc-365237), β-Tubulin (E7, Developmental Studies Hybridoma Bank), Histone H3 (Shilatifard Laboratory, no. 42), and Flag-M2 (Sigma, no. F1804).

Techniques: Staining, SDS Page, Control, Expressing, Protein-Protein interactions, Western Blot, Immunoprecipitation, Incubation