Journal: Oncotarget

Article Title: E2F1 interactions with hHR23A inhibit its degradation and promote DNA repair

doi: 10.18632/oncotarget.8362

Figure Lengend Snippet: A, B. Primary keratinocytes were transfected with vectors encoding V5-tagged E2F1 with or without FLAG-tagged hHR23A or HA- and GFP-tagged hHR23A and cultured for 24 h after transfection in Low Ca 2+ or in High Ca 2+ medium, to induce differentiation. Cell lysates were prepared and immunoprecipitated with anti-FLAG or anti-V5 antibodies, as indicated, or an irrelevant IgG. Immune complexes were resolved by denaturing gel electrophoresis, transferred to membranes, and the blots were probed with the indicated antibodies. Samples of lysates used for immunoprecipitation show expression levels of exogenous proteins. γ-Tubulin was used to normalize for protein loading, and the asterisks indicate a non-specific band. C. Lysates prepared from transfected keratinocytes as in (A, B), were immunoprecipitated with anti-E2F1 antibodies, to isolate endogenous and/or exogenous E2F1 immune complexes. Replicate lysate samples were also analyzed to show expression levels of endogenous and exogenous proteins. D. Bacterially produced GST, GST-E2F1, as well as His- and FLAG-tagged hHR23A (2 μg each) were used in immunoprecipitation experiments with anti-GST or anti-His antibodies, as indicated. The immune complexes were further analyzed by immunoblot, using anti-FLAG or anti-E2F1 antibodies. The lanes labelled “Input” contain 100 ng each of the indicated recombinant proteins.

Article Snippet: Primary mouse keratinocytes were isolated from 2 d-old CD-1 mice and cultured in Ca 2+ -free EMEM (06-174G, Lonza, Rockland, ME) supplemented with growth additives and 8% fetal bovine serum (FBS) pre-treated with Chelex resin, as described [ , ].

Techniques: Transfection, Cell Culture, Immunoprecipitation, Nucleic Acid Electrophoresis, Expressing, Produced, Western Blot, Recombinant

Journal: Oncotarget

Article Title: E2F1 interactions with hHR23A inhibit its degradation and promote DNA repair

doi: 10.18632/oncotarget.8362

Figure Lengend Snippet: A. Primary undifferentiated keratinocytes were transfected with vectors encoding V5-tagged E2F1 and HA-tagged ubiquitin, in the presence or absence of hHR23-encoding vectors, as indicated. The cells were cultured for 24 h in Low Ca 2+ (undifferentiated keratinocytes) or High Ca 2+ medium (differentiated keratinocytes) in the absence of MG132. Cell lysates were prepared and ubiquitylated proteins were immunoprecipitated with anti-HA antibodies. HA immune complexes were analyzed with anti-V5 antibodies, to detect ubiquitylated E2F1 in the immune complexes. The asterisk indicates a lower mobility non-specific band. B. Primary undifferentiated keratinocytes were transfected with a vector encoding V5-tagged E2F1 and either wild type (WT) ubiquitin, or a ubiquitin mutant lacking all Lys residues (K0), in the presence or absence of FLAG-tagged hHR23A. Keratinocytes were treated with vehicle (dimethylsulfoxide) or MG132 (10 μM) for 6 h, and cell lysates were prepared, immunoprecipitated with anti-HA antibodies and analyzed for the presence of ubiquitylated V5-E2F1, as in (A).

Article Snippet: Primary mouse keratinocytes were isolated from 2 d-old CD-1 mice and cultured in Ca 2+ -free EMEM (06-174G, Lonza, Rockland, ME) supplemented with growth additives and 8% fetal bovine serum (FBS) pre-treated with Chelex resin, as described [ , ].

Techniques: Transfection, Ubiquitin Proteomics, Cell Culture, Immunoprecipitation, Plasmid Preparation, Mutagenesis