Anti gapdh 6c5

HCV core 4a and core 4f genes were amplified by PCR from pCI/core-4aR, pCI/core-4fC plasmid (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6060129/) (accessed on 12 April 2022) and placed downstream of a transthyretin (TTR) liver-specific promoter [32 (link)]. Core 4a and core 4f genes was inserted into the StuI site of the pTTR1-ExV3 plasmid, and the transgenes were prepared by purifying the HindIII fragment containing TTR promoter and the core 4a/core 4f coding sequence. The TTR promoter was kindly provided by Dr. Iannis Talianidis, Institute of Molecular Biology and Biotechnology of FORTH in Crete, Heraklion, Greece. Transgenic CBA-C57BL/6 mice, which express liver-specifically core 4a (TTRcore4a) and core 4f (TTRcore4f) proteins, were generated in the Transgenesis Facility of the Biomedical Sciences Research Center “Alexander Fleming”, Vari, Greece. Mice were maintained under specific pathogen-free (SPF) conditions. Western blot using anti-GAPDH (6C5, Abcam, Cambridge, MA, USA) and anti-HCV core (C7-50, Abcam, Cambridge, MA, USA) antibodies was conducted according to the manufacturer’s general guidelines.

For immunoblot analysis, HEK293 T-REx drTPRM7 wild type or truncation mutant cells were induced for 16 h with 1 μg/mL tetracycline added to the media. Cells were harvested and dissolved in lysis buffer (10 mM Tris-HCl, 75 mM NaCl, 5% glycerol, 0.5% triton, 5 mM EDTA, 1 mM PMSF) containing protease inhibitors (104 mM AEBSF, 80 μM Aprotinin, 4 mM Bestatin, 1.4 mM E-64, 2 mM Leupeptin and 1.5 mM Pepstatin A) for 30 minutes at 4 °C. After incubation, the lysates were centrifuged at 12,000 rpm for 5 min. at 4 °C and the protein concentration of the lysates were measured using a protein assay reagent (Thermo Scientific, USA). Soluble lysates were boiled with NuPAGE LDS sample buffer and NuPAGE Reducing Agent (Invitrogen) at 95 °C for 8 minutes. Equal amounts of proteins (40 μg) were loaded and separated in a NuPAGE 4–12% gel (Invitrogen) then transferred to a PVDF membrane. Proteins were detected by immunoblotting with the antibodies anti-HA (3F10, Roche), anti-GAPDH (6C5, Abcam, UK) followed by treatment with horseradish peroxidase (HRP)-conjugated anti-rat or anti-mouse antibody (GE Healthcare, USA). GAPDH was evaluated as an internal control. The antibody-bound protein was visualized using ECL solution (Life Technologies, USA.)

Whole-cell protein lysates were prepared from exponentially growing cells. Cells were resuspended in 150μL PBS (Invitrogen) supplemented with protease inhibitor (Roche Complete Mini) and 150μL 2X Laemmli sample buffer (Sigma) supplemented with protease inhibitor was then added, mixed and boiled for 10 minutes at 100°C. Protein lysates were resolved by SDS-PAGE on Novex 4% Tris-Glycine gels (Invitrogen), transferred to nitrocellulose membranes (Invitrogen), and probed with primary and horseradish peroxidase-conjugated secondary antibodies. Membranes were blocked and probed in 4% cold water fish skin gelatin (Sigma). The primary antibodies used in this study are anti-Ki-67 (MIB-1, Santa Cruz), anti-GAPDH (6C5, Abcam), anti-cyclin D1 (2922, Cell Signaling) and anti-cyclin E (M-20, Santa Cruz).