ha cat no 3724s  (Cell Signaling Technology Inc)

Journal: Tumour Virus Research

Article Title: Oncolytic virus hijacks GOT1 and pyrimidinosomes to fuel pyrimidine synthesis for replication in tumor cells

doi: 10.1016/j.tvr.2026.200342

Figure Lengend Snippet: | NDV infection promotes the assembly of pyrimidinosome involving GOT1 and pyrimidine synthases. (A) H1299 cells were co-transfected with GFP-GOT1 and mCherry-UMPS for 24 h, followed by mock infection or NDV infection (MOI = 0.1, 1, 5, 10, or UV-NDV for 12 hpi). Cells were then analyzed using confocal microscopy. (B) H1299 cells were co-transfected with GFP-GOT1 and mCherry-UMPS for 24 h, followed by mock infection or NDV infection (MOI = 1 for 6, 12, and 18 hpi). Cells were then analyzed using confocal microscopy. (C) Immunofluorescence analysis (IFA) of mock-infected and NDV-infected A549 cells (MOI = 1, 12 hpi), stained for endogenous GOT1, UMPS, and DHODH, alongside the mitochondrial marker protein Tom 20. (D-F) H1299 cells were co-transfected with GFP-GOT1 and mCherry-UMPS (D), GFP-DHODH and mCherry-UMPS (E), or GFP-GOT1 and mCherry-DHODH (F) for 24 h, followed by mock infection or NDV infection (MOI = 1, 12 hpi). Cells were then analyzed using confocal microscopy. (G-H) HEK293T cells were mock-infected or infected with NDV (MOI = 1) for 12 h. The cells were lysed and subjected to immunoprecipitation (IP) using GOT1 (G) and UMPS (H) antibodies, alongside with anti-IgG antibody as a control, followed by WB with anti-CAD, -UMPS, -DHODH and -GOT1 antibodies. (I-J) H1299 cells were co-transfected with Flag-CAD and HA-GOT1 for 24 h, followed by mock infection or NDV infection (MOI = 1, 12 hpi). The cells were lysed and subjected to IP using anti-Flag (I) or anti-HA (J) magnetic beads, followed by WB with anti-Flag and anti-HA antibodies. (K) H1299 cells were transfected with GFP-GOT1, mCherry-UMPS, or GFP-DHODH for 24 h, followed by NDV infection (MOI = 1, 12 hpi). FRAP analysis was then performed using confocal microscopy.

Article Snippet: Anti-Flag Magnetic Beads (Cat# HY-K0207), anti-HA Magnetic Beads (Cat# HY-K0201), Mycophenolate (Cat# HY-B0421), Leflunomide (Cat# HY-B0083), AG 2037 (Cat# HY-14530), Aminooxyacetic acid hemihydrochloride (Cat# HY-107994) were purchased from MedChemExpress (MCE).

Techniques: Infection, Transfection, Confocal Microscopy, Immunofluorescence, Staining, Marker, Immunoprecipitation, Control, Magnetic Beads

Journal: Tumour Virus Research

Article Title: HPV18E6 and CDK5 virus-host interaction is a prospective therapeutic target for HPV-positive cervical cancer

doi: 10.1016/j.tvr.2026.200339

Figure Lengend Snippet: CDK5 forms a complex with E6 predominantly in the nucleus. (A) (i). The represented immunoblot image of CDK5 protein binding with 16E6/E7 and 18E6/E7 proteins. GST pull-down assay was performed by incubating the indicated purified GST fusion proteins with CDK5 protein. After extensive washing, the bound CDK5 protein was detected via Western blotting using an anti-CDK5 antibody. The immunoblot (IB) on the upper panel shows the interaction of CDK5 with GST-16 E6/E7 and GST-18 E6/E7, while the lower panel shows the Ponceau S stain of the blot. (ii) The bar graph shows the quantification of the relative level of CDK5 to GST empty protein indicated from 3 independent experiments (n = 3). Quantitation was performed using ImageJ software, and the statistical analysis was performed using Graphpad Prism 8. (B). Representatives immunoblot of Co-immunoprecipitation shows that CDK5 binds with 16E6 and 18E6. The HA-16E6, HA-18E6, and His-CDK5 were transfected into the HEK293 cells. After 24 h, the lysates from cells were analyzed by western blotting using anti-CDK5, anti-HA specific antibodies. Data were expressed as mean ± standard error of the mean (SEM, ∗: p < 0.05, ∗∗: p < 0.01, ∗∗∗: p < 0.005, ∗∗∗∗: p < 0.0001). (C). U-2 OS cells were transfected with pcDNA3.1: His-CDK5 (His-CDK5) and pcDNA3.1: HA-16E6 (HA-16E6) and HA-18E6 (HA-18E6) plasmids. The cells were fixed and incubated with primary antibodies (anti-His, anti-HA), followed by incubation with the relevant Alexa Fluor 568-conjugated anti-rabbit and Alexa Fluor 488-conjugated anti-mouse secondary antibodies. The cells were then counterstained with 4,6-diamidino-2-phenylindole (DAPI). The Z-stacking images for subcellular expression of CDK5 (Red) and E6 (Green) were examined using the Nikon fluorescence microscope. Cellular localization of CDK5 and E6 was visualized by a fluorescent microscope under 1000× magnification.

Article Snippet: The transferred membranes were incubated overnight at 4 °C with the following primary antibodies: monoclonal rabbit anti-HA (Cell Signaling Technology), monoclonal mouse anti-p53 (Santa Cruz), monoclonal mouse anti-HPV18E6 (Santa Cruz), monoclonal mouse anti-CDK5 (Santa Cruz), and monoclonal mouse anti-β-actin (Santa Cruz).

Techniques: Western Blot, Protein Binding, Pull Down Assay, Purification, Staining, Quantitation Assay, Software, Immunoprecipitation, Transfection, Incubation, Expressing, Fluorescence, Microscopy

Journal: Tumour Virus Research

Article Title: HPV18E6 and CDK5 virus-host interaction is a prospective therapeutic target for HPV-positive cervical cancer

doi: 10.1016/j.tvr.2026.200339

Figure Lengend Snippet: Elevation of CDK5 contributes to an increased steady-state level of E6. (A) (i). The CDK5 can stabilise the 18E6 protein. HEK 293 cells were transfected with pCDNA3.1: HA-18E6 and/or pcDNA3.1: His-CDK5 for 24 h and incubated with cycloheximide (CHX) at different time points as indicated. Total protein lysates were extracted from cells and subjected to Western blotting using the anti-HA, anti-CDK5, and β-actin (a loading control) antibodies. (ii). The curve shows the protein level of 18E6 with or without the overexpression of CDK5. The stability of 18E6 was analyzed by the one-phase exponential decay using GraphPad Prism 8. (B) (i-ii). CP681301 attenuated the stabilisation of the 18E6 protein. HeLa cells were treated with 0.6 μM CP681301 or DMSO for 24 h, and then incubated with cycloheximide (CHX) at different time points (0, 30, 60, 120 min). Total protein lysates were extracted from cells and subjected to Western blotting using the anti-18E6, anti-CDK5, and β-actin (a loading control) antibodies. (C). The curve shows the protein level of the treatment group and the non-treatment group. The stability of 18E6 was analyzed by the one-phase exponential decay using GraphPad Prism 8.

Article Snippet: The transferred membranes were incubated overnight at 4 °C with the following primary antibodies: monoclonal rabbit anti-HA (Cell Signaling Technology), monoclonal mouse anti-p53 (Santa Cruz), monoclonal mouse anti-HPV18E6 (Santa Cruz), monoclonal mouse anti-CDK5 (Santa Cruz), and monoclonal mouse anti-β-actin (Santa Cruz).

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