Mapping RNF187-P53 Protein Interactions

Immunoprecipitation was performed as described in a previous study [42 (link)]. Total MCF-7 cell lysates were precleared with rabbit IgG for 2 h and subsequently immunoprecipitated with an anti-RNF187 antibody (HPA030098, Sigma) overnight, while rabbit IgG (Santa Cruz) was used as the negative control. Bound proteins were analyzed by western blotting with an anti-P53 antibody (SC126, Santa Cruz). For the overexpression experiment, HEK293 cells were cotransfected with 5 μg of GFP-RNF187 plasmid (full-length RNF187 or domain deletion mutants) and 5 μg of P53 plasmid. Cell lysates were precleared with IgG and subsequently incubated with an anti-P53 (SC126, Santa Cruz) antibody, while mouse IgG was used as the negative control. Bound proteins were analyzed by western blotting with an anti-GFP antibody (AB290, Abcam). Accordingly, the GFP-P53 plasmid (full-length P53 or domain deletion mutants) was cotransfected with 5 μg of Myc-RNF187 plasmid in 10 cm dishes. Cell lysates were precleared with IgG and subsequently incubated with an anti-Myc (AB32, Abcam) antibody, while rabbit IgG was used as the negative control. Bound proteins were analyzed by western blotting.

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Li X., Niu Z., Sun C., Zhuo S., Yang H., Yang X., Liu Y., Yan C., Li Z., Cao Q., Ji G., Ding Y., Zhuang T, & Zhu J. (2022). Regulation of P53 signaling in breast cancer by the E3 ubiquitin ligase RNF187. Cell Death & Disease, 13(2), 149.

Publication 2022

HPA030098 SC126 AB290

Anti antibody Antibody Cell Deletion Dishes Hek293 cells Immunoprecipitation Mcf 7 cell Mouse Plasmid Proteins Rabbit

Corresponding Organization :

Other organizations : Xinxiang Medical University, First Affiliated Hospital of Zhengzhou University, Harvard Stem Cell Institute, Xinxiang University, Second Hospital of Shandong University

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Variable analysis

independent variables

Overexpression of GFP-RNF187 plasmid (full-length RNF187 or domain deletion mutants)
Overexpression of Myc-RNF187 plasmid
Overexpression of GFP-P53 plasmid (full-length P53 or domain deletion mutants)

dependent variables

Interaction between RNF187 and P53 proteins, as measured by co-immunoprecipitation and western blotting

control variables

Cell lines: MCF-7 and HEK293

positive controls

Rabbit IgG for preclearing cell lysates
Anti-P53 antibody (SC126, Santa Cruz) for immunoprecipitation

negative controls

Rabbit IgG for immunoprecipitation (negative control for RNF187-P53 interaction)
Mouse IgG for immunoprecipitation (negative control for GFP-P53 and Myc-RNF187 interaction)

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