Hpa030098

MCF-7 cells were fixed with 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.2% Triton X-100 for 5 min, and blocked with 5% BSA in PBS for 1 h. A rabbit anti-RNF187 (HPA030098, Sigma, 1:100) antibody and mouse anti-P53 monoclonal antibody (SC126, Santa Cruz, 1:100) were used as primary antibodies, followed by Alexa Fluor 647-conjugated (Invitrogen) anti-rabbit and FITC-conjugated anti-mouse antibodies (Jackson ImmunoResearch, West Grove, PA) as secondary antibodies. As negative controls, samples were incubated with secondary antibodies without the primary antibody incubation step. Images were acquired under conditions satisfying the Nyquist criterion using a Nikon A + laser scanning confocal microscope system with a 60X oil NA1.4 objective and pinhole size of 1.0 Airy unit. The acquired images were further processed and assembled using ImageJ.

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.