Journal: PLoS ONE

Article Title: Potent Anti-Tumor Effect Generated by a Novel Human Papillomavirus (HPV) Antagonist Peptide Reactivating the pRb/E2F Pathway

doi: 10.1371/journal.pone.0017734

Figure Lengend Snippet: (A) Competition between positive clones and pRb at concentrations indicated for binding to HPV16 E7. (B) His-pull down experiment. Lane 1, 293T cell lysates tested by Western blot directly; Lane 2, HPV16 E7 protein tested by Western blot directly; Lane 3, pRb pulled down by HPV16 E7-His-band resin from 293T cell lysates; Lane 4, pRb pulled down by HPV16 E7-His-band resin from 293T cell lysates in the presence of Pep-7. (C), Co-IP of HPV16 E7 and pRb. Cos7 cells co-transfected with His-pRb or YFP-HPV16E7 were treated with (lane 2) or without Pep-7 (lane 1 and 3). The cell lysates (left panel) or immunoprecipitates of pRb antibody (right panel, lane 1 and 2) or Flag antibody (right panel, lane 3) were blotted with HPV16 E7 or pRb antibodies; (D) half-life study of pRb with or without Pep-7 treatment. SiHa cells incubated with or without 80 µM Pep-7 were treated with 50 µg/ml cycloheximide for 12 h, and the cells were harvested for Western blotting at time points indicated; (E) phosphorylation of pRb assay in cells treated with or without Pep-7. SiHa cells were harvested and subjected to Western blotting probed with pRb and Phospho-Rb antibodies respectively, post 24 h incubation with or without 80 µM Pep-7. β-actin was used as loading control, and protein level was calculated from the band intensities of each proteins relative to that of β-actin ( * , p<0.02; ** , p<0.01).

Article Snippet: Mouse monoclonal to p130/Rb2 antibody was supplied from NeoMarkers For Lab Vision Corporation (Fremont, CA, USA). pRb Antibody Sampler Kit, including Phospho-pRb (Ser780), Phospho-pRb (Ser795) and Phospho-pRb (Ser807/811) antibodies and pRb (4H1) Mouse antibody, was supplied by Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Clone Assay, Binding Assay, Western Blot, Co-Immunoprecipitation Assay, Transfection, Incubation