n terminal ha tagged ubiquitin ub expression plasmids  (Addgene inc)

Journal: bioRxiv

Article Title: HCV infection induces ubiquitin-dependent degradation of LATS1, inactivating the Hippo pathway and upregulating transcription of the CYR61 and CTGF genes

doi: 10.1101/2025.04.08.647823

Figure Lengend Snippet: Huh-7.5 cells were infected with HCV J6/JFH1 at an MOI of 0.5 and co-transfected with pCAG-FLAG-LATS1 together with the indicated HA-tagged ubiquitin mutant plasmids. At 2 days post-transfection, cells were harvested and cell lysates were immunoprecipitated with anti-FLAG beads, followed by immunoblotting with the indicated antibodies. Input samples were also analyzed by immunoblotting with the indicated antibodies, and β-actin was used as a loading control.

Article Snippet: N-terminal HA-tagged ubiquitin (Ub) expression plasmids, including pRK5-HA−Ub-WT, pRK5-HA−Ub-K6, pRK5-HA−Ub-K11, pRK5-HA−Ub-K27, pRK5-HA−Ub-K29, pRK5-HA−Ub-K33, pRK5-HA−Ub-K48, and pRK5-HA−Ub-K63, were purchased from Addgene (Watertown, MA).

Techniques: Infection, Transfection, Mutagenesis, Immunoprecipitation, Western Blot, Control

Journal: bioRxiv

Article Title: HCV infection induces ubiquitin-dependent degradation of LATS1, inactivating the Hippo pathway and upregulating transcription of the CYR61 and CTGF genes

doi: 10.1101/2025.04.08.647823

Figure Lengend Snippet: (A) Huh-7.5 cells were co-transfected with pCAG-Myc-LATS1, pRK5-HA-Ubiquitin, together with either pCAG-FLAG-Itch WT or pCAG-FLAG-Itch C868A. At 2 days post-transfection, the cells were harvested. The cell lysates were immunoprecipitated with anti-c-Myc antibody-conjugated Sepharose A beads, followed by immunoblotting with the indicated antibodies. Input samples were also analyzed by immunoblotting. β-actin was used as a loading control. (B) Huh-7.5 cells were co-transfected with pCAG-Myc-LATS1, pRK5-HA−Ubiquitin, together with either pCAG-FLAG−WWP1 WT or pCAG-FLAG−WWP1 C890A. At 2 days post-transfection, cells were harvested. The cell lysates were immunoprecipitated with anti-c-Myc antibody−conjugated Sepharose A beads, followed by immunoblotting with the indicated antibodies. Input samples were also immunoblotted, with β-actin serving as a loading control. (C) Huh-7.5 cells were infected with HCV J6/JFH1 at an MOI of 1 and co-transfected with pCAG-Myc-LATS1 and pRK5-HA−Ubiquitin, together with either pCAG-FLAG-Itch or pCAG-FLAG−WWP1. Cell lysates were immunoprecipitated using anti-c-Myc antibody−conjugated Sepharose A beads, followed by immunoblotting with the indicated antibodies. Input samples were also analyzed. β-actin was used as a loading control. (D) Huh-7.5 cells (3×10 5 cells per well in a 12-well plate) were transfected with 48 pmol of either control siRNA or Itch-specific siRNA. After 24 h, the cells were infected with HCV J6/JFH1 at an MOI of 2 and harvested at 2 days post-infection. The samples were analyzed by immunoblotting with the indicated antibodies. β-actin was used as a loading control. (E) Huh-7.5 cells (3×10 5 cells per well in a 12-well plate) were transfected with 48 pmol of either control siRNA or WWP1-specific siRNA. At 24 h after siRNA-transfection, the cells were infected with HCV J6/JFH1 at an MOI of 2. Cells were harvested at 2 days post-infection, and the samples were subjected to immunoblotting with the indicated antibodies. β-actin was used as a loading control.

Article Snippet: N-terminal HA-tagged ubiquitin (Ub) expression plasmids, including pRK5-HA−Ub-WT, pRK5-HA−Ub-K6, pRK5-HA−Ub-K11, pRK5-HA−Ub-K27, pRK5-HA−Ub-K29, pRK5-HA−Ub-K33, pRK5-HA−Ub-K48, and pRK5-HA−Ub-K63, were purchased from Addgene (Watertown, MA).

Techniques: Transfection, Immunoprecipitation, Western Blot, Control, Infection

Journal: bioRxiv

Article Title: HCV infection induces ubiquitin-dependent degradation of LATS1, inactivating the Hippo pathway and upregulating transcription of the CYR61 and CTGF genes

doi: 10.1101/2025.04.08.647823

Figure Lengend Snippet: Huh-7.5 cells were infected with HCV J6/JFH1 at an MOI of 1, then co-transfected with pCAG-Myc-LATS1 and pRK5-HA-ubiquitin, together with pCAG-FLAG-Itch. After 2 days, the cells were harvested with or without pretreatment with JNK inhibitor SP6000125 (30 µM for 30 h), and the lysates were immunoprecipitated with anti-c-Myc antibody−conjugated Sepharose A beads, followed by immunoblotting with the indicated antibodies. Input samples were also analyzed by immunoblotting. β-actin was used as a loading control.

Article Snippet: N-terminal HA-tagged ubiquitin (Ub) expression plasmids, including pRK5-HA−Ub-WT, pRK5-HA−Ub-K6, pRK5-HA−Ub-K11, pRK5-HA−Ub-K27, pRK5-HA−Ub-K29, pRK5-HA−Ub-K33, pRK5-HA−Ub-K48, and pRK5-HA−Ub-K63, were purchased from Addgene (Watertown, MA).

Techniques: Infection, Transfection, Immunoprecipitation, Western Blot, Control

Journal: bioRxiv

Article Title: HCV infection induces ubiquitin-dependent degradation of LATS1, inactivating the Hippo pathway and upregulating transcription of the CYR61 and CTGF genes

doi: 10.1101/2025.04.08.647823

Figure Lengend Snippet: Under normal conditions, the Hippo pathway remains predominantly active, with LATS1 phosphorylating YAP1, thereby retaining YAP1 in the cytoplasm and preventing its nuclear entry. HCV infection activates the ROS/JNK/Itch signalling pathway, which is crucial for HCV particle release. Activation of the ROS/JNK/Itch signalling pathway leads to the phosphorylation and enhanced activity of Itch ubiquitin ligase. Activated Itch promotes the ubiquitin-dependent proteasomal degradation of LATS1 protein, leading to the Hippo pathway inactivation. As a result, YAP1 translocates from the cytoplasm to the nucleus and upregulates the transcription of CYR61 and CTGF genes, contributing to HCV-related pathogenesis.

Article Snippet: N-terminal HA-tagged ubiquitin (Ub) expression plasmids, including pRK5-HA−Ub-WT, pRK5-HA−Ub-K6, pRK5-HA−Ub-K11, pRK5-HA−Ub-K27, pRK5-HA−Ub-K29, pRK5-HA−Ub-K33, pRK5-HA−Ub-K48, and pRK5-HA−Ub-K63, were purchased from Addgene (Watertown, MA).

Techniques: Infection, Activation Assay, Activity Assay

Journal: Journal of Virology

Article Title: HSV-1 UL56 protein recruits cellular NEDD4-family ubiquitin ligases to suppress CD1d expression and NKT cell function

doi: 10.1128/jvi.02140-24

Figure Lengend Snippet: UL56 interacts with NEDD4-family E3 ubiquitin ligases and may recruit to suppress CD1d expression. ( A ) Cellular proteins associated with HSV-1 UL56 protein were purified from cell lysates of transfected 293T.CD1d cells expressing GST-fused UL56 (1–215) by GST-pulldown. The high-molecular-weight proteins co-purified with GST-UL56 protein were subjected to mass spectrometry analysis to identify these proteins. Cells expressing GST protein were used as a control. ( B–D ) Individual members of FLAG-tagged NEDD4-family E3 ubiquitin ligases were co-transfected with pTracer plasmid in 293T.CD1d cells. Transfected cells were subjected to western blotting to verify the expression of individual E3 ubiquitin ligases ( B ) or stained with anti-CD1d antibody, CD1d.51.1 and cell surface CD1d expression was analyzed by flow cytometry ( C, D ). Transfected and untransfected cells were gated as GFP-positive and GFP-negative cells, respectively. ( D ) Quantitation of CD1d downregulation upon expression of NEDD4-family E3 ubiquitin ligases. The relative CD1d MFI was calculated as (CD1d MFI of CD1d in GFP-positive cells)/(CD1d MFI in GFP-negative cells). The average and standard deviations of relative CD1d MFI were calculated from three independently repeated experiments and plotted. Statistical analysis by one-way ANOVA with Duncan post hoc multiple comparisons. n.s., not significant. * P < 0.05, ** P < 0.01.

Article Snippet: The plasmid constructs expressing NEDD4 ubiquitin ligase family protein constructs expressed in the pCIneo vector (Promega) were generously provided by Dr. Wesley Sundquist at University of Utah and reported previously ( ).

Techniques: Ubiquitin Proteomics, Expressing, Purification, Transfection, High Molecular Weight, Mass Spectrometry, Control, Plasmid Preparation, Western Blot, Staining, Flow Cytometry, Quantitation Assay

Journal: Journal of Virology

Article Title: HSV-1 UL56 protein recruits cellular NEDD4-family ubiquitin ligases to suppress CD1d expression and NKT cell function

doi: 10.1128/jvi.02140-24

Figure Lengend Snippet: NEDD4L is the major NEDD4-family ubiquitin ligase interacting with UL56 and cooperating with UL56 to suppress CD1d expression. ( A, B ) 293T.CD1d cells were transfected with plasmids expressing UL56 alone or together with FLAG-tagged NEDD4L and subjected to co-immunoprecipitation with anti-UL56 antibodies or anti-FLAG antibodies. Immunoprecipitates were western blotted with anti-FLAG or anti-UL56 antibodies, respectively. Cellular Grp94 protein was used as a loading control. ( C–E ) 293T.CD1d were transfected with either pTracer alone or together with plasmids to express UL56 alone, NEDD4L alone, UL56 plus NEDD4L, or KSHV K5 proteins and subjected to western blotting ( C ) or cell surface staining for CD1d expression ( D ). Transfected and untransfected cells were gated as GFP-positive and GFP-negative cells, respectively. ( E ) Quantitation of CD1d downregulation. The relative CD1d MFI was calculated as (CD1d MFI of CD1d in GFP-positive cells)/(CD1d MFI in GFP-negative cells). The average and standard deviations of relative CD1d MFI were calculated from three independently repeated experiments and plotted. The experiments were repeated three times and results were summarized. Statistical analysis by one-way ANOVA with Duncan post hoc multiple comparisons. n.s., not significant. * P < 0.05, ** P < 0.01.

Article Snippet: The plasmid constructs expressing NEDD4 ubiquitin ligase family protein constructs expressed in the pCIneo vector (Promega) were generously provided by Dr. Wesley Sundquist at University of Utah and reported previously ( ).

Techniques: Ubiquitin Proteomics, Expressing, Transfection, Immunoprecipitation, Western Blot, Control, Staining, Quantitation Assay