Journal: Journal of Biological Chemistry

Article Title: Isolation and Characterization of a Novel H1.2 Complex That Acts as a Repressor of p53-mediated Transcription

doi: 10.1074/jbc.m708205200

Figure Lengend Snippet: FIGURE 4. Direct interaction of H1.2 with p53. A, p53 interaction with H1.2 in vitro. GST-H1.2 mutants (lanes 1–4) or GST-p53 mutants (lanes 5–7) were analyzed by SDS-PAGE and Coomassie staining analysis. For interaction studies, GST-H1.2 mutants and GST-p53 mutants were incubated with FLAG-tagged p53 and His-tagged H1.2, respectively. After washing, binding of p53 and H1.2 was analyzed by Western blot analysis with anti-FLAG or anti-His antibody. Lane 1, GST-H1.2 full length (FL; amino acids 1–213); lane 2, GST-H1.2 NT (amino acids 1–34); lane 3, GST-H1.2 globular domain (GD; amino acids 35–109); lane 4, GST-H1.2 CT (amino acids 110–213); lane 5, GST-p53 NT (amino acids 1–83); lane 6, GST-p53 DNA binding domain (DBD; amino acids 120–290); lane 7, GST-p53 CT (amino acids 290–393); lanes 8 and 14, 10% input of FLAG-p53 and His-H1.2; lanes 9 and 15, GST control; lanes 10–13, p53 bound to GST-H1.2 mutants; lanes 16–18, H1.2 bound to GST-p53 mutants. B, p53 interaction with H1.2 complex in vitro. GST (lane 1) or GST-p53 full length (lane 2) was incubated with H1.2 complex, and pulldown fractions were analyzed by Western blot analysis using the indicated antibodies. C, p53 interaction with H1.2 in vivo. H1.2 and p53 were expressedin293Tcellsandimmunoprecipitatedusinganti-FLAGandanti-Xpressantibodiesasindicated(lanes1–6).Similarexperimentswerealsoperformed after expression of p53 and H1.2 deletion mutants as described under “Experimental Procedures” (lanes 10–21). Lanes 1 and 4, p53-only expression; lanes 2 and 5, H1.2-only expression; lanes 3 and 6, p53 and H1.2 co-expression; lanes 7–9, expressed H1.2 mutants in whole cell lysates; lanes 10–12, H1.2-only controls; lanes 16–18,p53-onlycontrols;lanes13–15and19–21,H1.2mutantsandp53mutantsco-expressions.TheasteriskindicatesanonspecificbandcontainingIgGlightchain. D, mutual interaction of endogenous p53 and H1.2. Whole cell extracts from 293T cells were immunoprecipitated with anti-p53 antibody (DO-1) and analyzed by Western blotting with anti-H1.2 and anti-p53 antibodies as indicated. Lane 1, whole cell lysate; lane 2, control IgG; lane 3, anti-p53 precipitates. E, p53 interaction with H1.2 complex in vivo. 293T cells were transfected with FLAG-tagged p53 and Xpress-tagged H1.2-expressing plasmids, and cell lysates were prepared 2 days after transfection. Lysates were immunoprecipitated with anti-FLAG and analyzed by Western blot analysis using the indicated antibodies.Lane 1, whole cell lysate;lane 2, control IgG; lane 3, anti-FLAG precipitates. IP, immunoprecipitate; Xp, Xpress; f, FLAG.

Article Snippet: For co-immunoprecipitation of endogenous proteins, 293T cell lysates were immunoprecipitated with anti-p53 monoclonal antibody (DO-1, Santa Cruz Biotechnology, Inc.) followed by immunoblotting with antibodies against H1.2 (Abcam) and p53.

Techniques: In Vitro, SDS Page, Staining, Incubation, Binding Assay, Western Blot, Control, In Vivo, Expressing, Immunoprecipitation, Transfection