RYBP stabilizes p53. (A) U2OS, A549, HCT116 and PC3 cells were transfected with a Myc-RYBP plasmid for 24 h. Immunoblotting was used to assess the MDM2, p53 and p21^WAF1 protein levels. (B) U2OS or PC3 cells were transfected with 100 nM of control or RYBP siRNA pool for 72 h. MDM2, p53 and RYBP proteins in whole lysates were detected by immunoblotting. (C) U2OS cells were transfected with GFP vector, or deletion mutants of GFP-RYBP (4 μg) for 24 h. The lysates were cleared, and SDS–PAGE was used to detect MDM2 and p53. (D,E) A549 cells were transfected with a CMV vector or CMV-RYBP (4 μg) for 24 h followed by exposure to CHX (10 μg/ml) for the indicated times. (D) Cell lysates were collected and detected as above. (E) The intensity of the p53 bands was analysed by densitometry (Bio-Rad Model GS-670 Imaging Densitometer; Bio-Rad, Hercules, CA, USA). The relative densitometry at each time point is expressed as a percentage of the density at time 0 h after normalization to the corresponding β-actin level. CHX, cycloheximide; CMV, cytomegalovirus; GFP, green fluorescent protein; MDM2, mouse double minute 2; RYBP, RING1- and YY1-binding protein; SDS–PAGE, SDS–polyacrylamide gel electrophoresis; siRNA, small interfering RNA.

RYPB regulates the activity of p53. (A) U2OS cells were transfected with siControl or siRYBP pool (100 nM) for 72 h, or transfected with a CMV or CMV-RYBP vector (4 μg) for 24 h. The total RNA was then extracted and qRT–PCR was carried out to detect the relative messenger RNA (mRNA) levels of CDKN1A (p21) and BAX; ^*P<0.05. (B) HCT116 cells with or without p53 were transfected with a vector expressing either GFP or GFP-RYBP for 24 h, and then the GFP-positive populations were examined for DNA content (upper panels). A plot of the percentages of each phase of the cell cycle in various cells following RYBP treatment is included in the lower left panel. Analyses of protein lysates from the same transfected cells are shown in the lower right panel. (C) U2OS cells were exposed either to a control or to an RYBP siRNA pool (100 nM) for 48 h. The cells were then exposed to 1.0 μM etoposide for an additional 24 h, and the expression of target proteins was examined by immunoblotting. BAX, Bcl2-associated X protein; CDKN1A, cyclin-dependent kinase inhibitor 1A; CMV, cytomegalovirus; GFP, green fluorescent protein; qRT–PCR, quantitative reverse transcription–PCR; RYBP, RING1- and YY1-binding protein; si, small interfering.

RYBP binds to MDM2. (A) COS7 cells (5 × 10⁵ cells per well) were transfected with 10 μg of T7-MDM2, Myc-RYBP or both for 24 h. The lysates were immunoprecipitated (IP) with T7 (left panel) or Myc (middle panel) antibodies or directly immunoblotted (IB) with T7 and Myc antibodies (right panel). (B) GST, GST-MDM2 or GST-RYBP proteins were incubated with in vitro-translated HA-RYBP or Myc-MDM2 at 4°C overnight. The bound proteins were resolved on SDS–PAGE and detected by HA (left panel) or MDM2 antibodies (right panel). (C) U2OS cells were lysed in NP-40 lysis buffer, and the lysates were immunoprecipitated with mouse IgG or MDM2 2A10 (left panel), rabbit IgG or rabbit RYBP antibodies (right panel) at 4°C overnight. Protein G beads were then added to the mixture and rotated for another 1 h at 4°C. After washing with NP-40 buffer, the precipitates were separated by SDS–PAGE and the target proteins were detected with MDM2 2A10, p53 DO-1 or RYBP antibodies. DO-1, antibody against p53; GST, glutathione S-transferase; HA, haemagglutinin; MDM2, mouse double minute 2; RYBP, RING1- and YY1-binding protein; SDS–PAGE, SDS–polyacrylamide gel electrophoresis.

RYBP inhibits MDM2-mediated ubiquitination of p53. (A) U2OS cells were transfected with plasmids expressing a GFP vector or GFP-RYBP (10 μg) for 24 h. The cell lysates were immunoprecipitated (IP) with p53 FL393 or MDM2 H221, and the MDM2 and p53 protein levels were determined using MDM2 2A10 or p53 DO-1. (B) U2OS cells were transfected with either the control or RYBP siRNA pools (100 nM) for 72 h. Immunoprecipitation was accomplished with the p53 FL393 antibody, and the corresponding proteins were detected by immunoblotting. (C,D) A549 cells were transfected with His-Ub, HA-p53, T7-MDM2 and Myc-RYBP as indicated in the figures. At 24 h after transfection, the cells were collected and divided into two fractions. One was used directly for Western blot analysis. The other was lysed with Ni-NTA lysis buffer, and the lysates were purified using Ni-NTA agarose beads and immunoblotted with corresponding antibodies. (E) A549 cells were transfected with His-Ub and with increasing doses of GFP-RYBP, or a mutant of GFP-RYBP lacking the MDM2-binding domain for 24 h. The same strategy as described in Fig. 4D was used to analyse the endogenous ubiquitination of MDM2. (F) U2OS cells were transfected with combinations of plasmids, including the MDM2 mutant lacking E3 ligase activity (C464A), as indicated, for 24 h. Target proteins in the whole-cell lysates were detected by immunoblotting. CHX, cycloheximide; DO-1, antibody against p53; GFP, green fluorescent protein; HA, haemagglutinin; MDM2, mouse double minute 2; Ni-NTA, nickel-nitrilotriacetic acid; RYBP, RING1- and YY1-binding protein; siRNA; small interfering RNA; Ub, ubiquitin.

RYBP is downregulated in cancer tissues compared with matched normal tissues. (A) Proteins were extracted from adjacent normal and malignant liver and lung tissues, and immunoblotting was used to detect the expression of RYBP and β-actin. (B) Immunohistochemical analyses were performed to determine the expression of RYBP in tumour and normal adjacent tissue samples. Representative pictures from liver and lung tissues were selected to show the alterations in the expression of RYBP. C, control tissue; RYBP, RING1- and YY1-binding protein; T, tumor tissue.