monoclonal rabbit anti ha  (Cell Signaling Technology Inc)

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

Journal: mBio

Article Title: Streptococcus mitis bacteriocins drive contact-dependent lysis of S. pneumoniae facilitating transformation in multispecies environments

doi: 10.1128/mbio.02716-25

Figure Lengend Snippet: S. mitis bacteriocins CabAB mediate contact-dependent growth inhibition of S. pneumoniae . Experiments were done using S. mitis G22 Δ blp4 Δ blp1 and S. pneumoniae D39. The bacteriocin clusters blp4 and blp1 were first deleted from the genome of S. mitis G22, as they have anti-pneumococcal activity , which could obscure CabAB-mediated effects. ( A ) Proportion of S. mitis and S. pneumoniae in dual-species biofilm grown for 24 h in competence-permissive conditions in 24-well plates. S. mitis mutant p( cab + ) contains the native cabABC copy and an additional copy under constitutive regulation (promoter p23 ) in a multicopy plasmid. ( B ) S. mitis was grown in competence-permissive conditions. Cell-free supernatants (CFS) were recovered, and cell pellets were boiled. CFS and heat-killed cells were added to S. pneumoniae cultures, and growth was monitored. ( C ) Bacterial loads of S. pneumoniae grown in the presence of S. mitis in direct contact (left) or separated with a 0.4 μm pore transwell membrane (right). Biofilms were grown in 12-well plates. As the surface area available for cell attachment is higher in 12-well plates than in 24-well plates, we hypothesize that this contributed to the observed differences in maximum cell counts for D39. ( D ) Corresponding bacterial loads of S. mitis G22 in experiment ( C ). ( E ) Representative images of CabA-HA and CabB-HA immunodetection on the surface of live competent S. mitis cells containing the native cabABC (negative control), cabA-HA cabBC, or cabA cabB-HA cabC genotypes. S. mitis cultures were induced with CSP G22 , incubated with a primary anti-HA rabbit antibody, and then with an Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody. Cells were stained with FM4-64 (membrane dye) and DAPI (nucleic acid dye). At least 10 independent fields were detected per experiment/condition. Experiments were repeated at least three times, independently. The ratio paired t -test was used to compare bacterial proportions in dual-species biofilms. The two-tailed unpaired Mann-Whitney U test was used to compare bacterial loads. ** P < 0.01. *** P < 0.001. ns not significant.

Article Snippet: Blocking was performed in PBS with 5% bovine serum albumin (BSA) for 1 h. Cells were pelleted and resuspended in 100 μL of primary rabbit monoclonal anti-HA antibody (clone C29F4; Cell Signaling Technology) diluted 1:100 in PBS with 1% BSA, and incubated for 1 h. Cells were washed twice in PBS with 0.1% Tween 20, resuspended in 100 μL of Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (Invitrogen) diluted 1:500 in PBS with 1% BSA, and incubated 1 h protected from light.

Techniques: Inhibition, Activity Assay, Mutagenesis, Plasmid Preparation, Membrane, Cell Attachment Assay, Immunodetection, Negative Control, Incubation, Staining, Two Tailed Test, MANN-WHITNEY