ha ub k48 plasmid  (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: (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