Journal: Biomolecules

Article Title: Ribosomal Protein S4 X-Linked as a Novel Modulator of MDM2 Stability by Suppressing MDM2 Auto-Ubiquitination and SCF Complex-Mediated Ubiquitination

doi: 10.3390/biom14080885

Figure Lengend Snippet: RPS4X interacts with MDM2 in the nucleoplasm. ( A ) HEK-293T cells were co-transfected with 3 µg of plasmid expressing V5-MDM2 with (+) or without (−) 3 µg of plasmid expressing FLAG-RPS4X. After 36 h, the cell extracts were prepared and subjected to immunoprecipitation (IP) using anti-FLAG antibody, followed by immunoblotting (IB) with anti-V5 and anti-FLAG antibodies. Total protein levels in whole-cell lysates (WCLs) were analyzed by IB using anti-FLAG, anti-V5, and anti-β-actin antibodies. ( B ) HeLa cell extracts were prepared and subjected to IP using control rabbit IgG or anti-RPS4X antibodies, followed by IB with anti-MDM2 and anti-RPS4X antibodies. Total protein levels in WCLs were analyzed by IB using anti-MDM2, anti-RPS4X, and anti-β-actin antibodies. ( C ) HeLa cells were transfected with 1 µg of plasmid expressing AcGFP-MDM2 or mCherry-RPS4X, respectively ( top panels ), and were co-transfected with 1 µg of plasmid expressing AcGFP-MDM2 and mCherry-RPS4X ( bottom panels ). After 24 h, the cells were counterstained with Hoechst 33342 (Hoechst) to detect the nuclei and visualized using a fluorescence microscope. Scale bars represent 20 µm. RPS4X, ribosomal protein S4X-linked; MDM2, mouse double minute 2. Please see the original images of in .

Article Snippet: Mouse anti-β-actin (MBL) antibody, anti-MDM2 antibody (HDM2-323; Santa Cruz Biotechnology), anti-RPS4X antibody (Proteintech, Rosemont, IL, USA), and control rabbit IgG (561; MBL) were also purchased.

Techniques: Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Western Blot, Control, Fluorescence, Microscopy

Journal: Biomolecules

Article Title: Ribosomal Protein S4 X-Linked as a Novel Modulator of MDM2 Stability by Suppressing MDM2 Auto-Ubiquitination and SCF Complex-Mediated Ubiquitination

doi: 10.3390/biom14080885

Figure Lengend Snippet: Determination of binding domains between RPS4X and MDM2. ( A ) Schematic diagram of full-length MDM2 (MDM2-full) and deletion mutants (MDM2-A, -B, and -C) used in this study. Characteristic domains of MDM2 are indicated as follows: black box, p53 binding domain; gray box, acidic domain; dot box, Zinc-finger domain; stripe box, RING-finger domain; a.a., amino acid. ( B ) HEK-293T cells were co-transfected with 3 µg of plasmid expressing Myc-RPS4X with (+) or without (−) 3 µg of plasmid expressing FLAG-MDM2-full, -MDM2-A, -MDM2-B, or -MDM2-C. After 36 h, the cell extracts were prepared and subjected to immunoprecipitation (IP) using anti-FLAG antibody, followed by immunoblotting (IB) with anti-Myc and anti-FLAG antibodies. Total protein levels in whole-cell lysates (WCLs) were analyzed by IB using anti-Myc, anti-FLAG, and anti-β-actin antibodies. ( C ) Schematic diagram of full-length RPS4X (RPS4X-full) and deletion mutants (RPS4X-N and -C) used in this study. a.a., amino acid. ( D ) HEK-293T cells were co-transfected with 2 µg of plasmid expressing V5-MDM2 with (+) or without (−) 2 µg of plasmid expressing Myc-RPS4X-full, -RPS4X-N, or -RPS4X-C. After 36 h, the cell extracts were prepared and subjected to IP using anti-Myc antibody, followed by IB with anti-V5 and anti-Myc antibodies. Total protein levels in WCLs were analyzed by IB using anti-V5, anti-Myc, and anti-β-actin antibodies. RPS4X, ribosomal protein S4X-linked; MDM2, mouse double minute 2. Please see the original images of in .

Article Snippet: Mouse anti-β-actin (MBL) antibody, anti-MDM2 antibody (HDM2-323; Santa Cruz Biotechnology), anti-RPS4X antibody (Proteintech, Rosemont, IL, USA), and control rabbit IgG (561; MBL) were also purchased.

Techniques: Binding Assay, Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Western Blot

Journal: Biomolecules

Article Title: Ribosomal Protein S4 X-Linked as a Novel Modulator of MDM2 Stability by Suppressing MDM2 Auto-Ubiquitination and SCF Complex-Mediated Ubiquitination

doi: 10.3390/biom14080885

Figure Lengend Snippet: MDM2 homodimer formation and MDM2 ubiquitination are suppressed by RPS4X. ( A ) HEK-293T cells were co-transfected with 1 µg of plasmid expressing Myc-MDM2 with (+) or without (−) 1 µg of plasmid expressing FLAG-MDM2 and 3 µg of plasmid expressing HA-RPS4X. After 24 h, the cells were treated with 20 µM MG132 (a proteasome inhibitor) for 16 h. The cell extracts were prepared and subjected to immunoprecipitation (IP) using anti-FLAG antibody, followed by immunoblotting (IB) with anti-Myc and anti-FLAG antibodies. Total protein levels in whole-cell lysates (WCLs) were analyzed by IB using anti-Myc, anti-FLAG, anti-HA, and anti-β-actin antibodies. ( B ) HEK-293T cells were co-transfected with 2 µg of plasmid expressing FLAG-MDM2 with (+) or without (−) 1 µg of plasmid expressing HA-ubiquitin (Ub) or Myc-RPS4X. After 24 h, the cells were treated with 40 µM MG132 (a proteasome inhibitor) for 16 h. The cell extracts were prepared and subjected to IP using anti-FLAG antibody, followed by IB with anti-HA and anti-FLAG antibodies. Total protein levels in WCLs were analyzed by IB using anti-FLAG, anti-HA, anti-Myc, and anti-β-actin antibodies. RPS4X, ribosomal protein S4X-linked; MDM2, mouse double minute 2. Please see the original images of in .

Article Snippet: Mouse anti-β-actin (MBL) antibody, anti-MDM2 antibody (HDM2-323; Santa Cruz Biotechnology), anti-RPS4X antibody (Proteintech, Rosemont, IL, USA), and control rabbit IgG (561; MBL) were also purchased.

Techniques: Ubiquitin Proteomics, Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Western Blot

Journal: Biomolecules

Article Title: Ribosomal Protein S4 X-Linked as a Novel Modulator of MDM2 Stability by Suppressing MDM2 Auto-Ubiquitination and SCF Complex-Mediated Ubiquitination

doi: 10.3390/biom14080885

Figure Lengend Snippet: RPS4X binds to Cullin1 and inhibits the interaction between MDM2 and Cullin1. ( A ) HEK-293T cells were co-transfected with 2 µg of plasmid expressing V5-MDM2 with (+) or without (−) 2 µg of plasmid expressing Myc-Cullin1 or HA-RPS4X. After 36 h, the cell extracts were prepared and subjected to immunoprecipitation (IP) using anti-Myc antibody, followed by immunoblotting (IB) with anti-V5 and anti-Myc antibodies. Total protein levels in whole-cell lysates (WCLs) were analyzed by IB using anti-V5, anti-Myc, anti-HA, and anti-β-actin antibodies. ( B ) HEK-293T cells were co-transfected with 2 µg of plasmid expressing Myc-Cullin1 with (+) or without (−) 2 µg of plasmid expressing FLAG-RPS4X. After 36 h, the cell extracts were prepared and subjected to IP using anti-FLAG antibody, followed by IB with anti-Myc and anti-FLAG antibodies. Total protein levels in WCLs were analyzed by IB using anti-Myc, anti-FLAG, and anti-β-actin antibodies. ( C ) HEK-293T cells were co-transfected with 2 µg of plasmid expressing FLAG-Cullin1 with (+) or without (−) 2 µg of plasmid expressing Myc-RPS4X-full, -RPS4X-N, or -RPS4X-C. After 36 h, the cell extracts were prepared and subjected to IP using the anti-Myc antibody, followed by IB with anti-FLAG and anti-Myc antibodies. Total protein levels in WCLs were analyzed by IB using the anti-FLAG, anti-Myc, and anti-β-actin antibodies. ( D ) Schematic diagram of the full-length Cullin1 (Cullin1-full) and deletion mutants (Cullin1-A, -B, and -C) used in this study. Characteristic domains of Cullin1 are indicated as follows: gray box, Skp1 binding domain; horizontal stripes box, Rbx1 binding domain; a.a., amino acid. ( E ) HEK-293T cells were co-transfected with 2 µg of plasmid expressing FLAG-RPS4X with (+) or without (−) 2 µg of plasmid expressing Myc-Cullin1-full, -Cullin1-A, -Cullin1-B, or -Cullin1-C. After 36 h, the cell extracts were prepared and subjected to IP using anti-Myc antibody, followed by IB with anti-FLAG and anti-Myc antibodies. Total protein levels in WCL were analyzed by IB using anti-FLAG, anti-Myc, and anti-β-actin antibodies. RPS4X, ribosomal protein S4X-linked; MDM2, mouse double minute 2. Please see the original images of in .

Article Snippet: Mouse anti-β-actin (MBL) antibody, anti-MDM2 antibody (HDM2-323; Santa Cruz Biotechnology), anti-RPS4X antibody (Proteintech, Rosemont, IL, USA), and control rabbit IgG (561; MBL) were also purchased.

Techniques: Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Western Blot, Binding Assay

Journal: Biomolecules

Article Title: Ribosomal Protein S4 X-Linked as a Novel Modulator of MDM2 Stability by Suppressing MDM2 Auto-Ubiquitination and SCF Complex-Mediated Ubiquitination

doi: 10.3390/biom14080885

Figure Lengend Snippet: MDM2 stability is enhanced by RPS4X. ( A ) HEK-293T cells were co-transfected with 0.5 µg of plasmid expressing Myc-MDM2 and FLAG-Cullin1 with (+) or without (−) 3 µg of plasmid expressing FLAG-RPS4X. After 24 h, the cells were treated with 100 µg/mL cycloheximide (protein synthesis inhibitor) and chased for the indicated time intervals. The cell lysates were subjected to SDS-PAGE followed by immunoblotting (IB) with anti-Myc antibody, and the MDM2 protein levels are shown. The intensity of each band was quantified by densitometry using ImageJ v1.43 software and graphed. ( B ) HEK-293T cells were co-transfected with 3 μg of plasmid expressing V5-MDM2 with (+) or without (−) 3 μg of plasmid expressing Myc-RPS4X. After 36 h, the cell extracts were subjected to SDS-PAGE, followed by IB with anti-V5, anti-Myc, and anti-β-actin antibodies. RPS4X, ribosomal protein S4X-linked; MDM2, mouse double minute 2. Please see the original images of in .

Article Snippet: Mouse anti-β-actin (MBL) antibody, anti-MDM2 antibody (HDM2-323; Santa Cruz Biotechnology), anti-RPS4X antibody (Proteintech, Rosemont, IL, USA), and control rabbit IgG (561; MBL) were also purchased.

Techniques: Transfection, Plasmid Preparation, Expressing, SDS Page, Western Blot, Software