anti rps4x antibody  (Proteintech)

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: Figure 1. 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)

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

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: Figure 2. 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 Figure 2 in Figure S2.

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: Figure 3. 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 sub- jected 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 Figure 3 in Figure S3.

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: Figure 4. 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

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

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: Figure 5. 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

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

Journal: Cell reports

Article Title: Early developmental deletion of forebrain Ank2 causes seizure-related phenotypes by reshaping the synaptic proteome

doi: 10.1016/j.celrep.2023.112784

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-RPS4X , Proteintech , Cat# 14799–1-AP; RRID: AB 2238567.

Techniques: Virus, Recombinant, Bicinchoninic Acid Protein Assay, Mass Spectrometry, Software

Journal: The EMBO Journal

Article Title: eIF3 mRNA selectivity profiling reveals eIF3k as a cancer‐relevant regulator of ribosome content

doi: 10.15252/embj.2022112362

Figure Lengend Snippet: Lysates of eIF3k‐mAID cells exposed to DMSO or IAA for 12 h were separated by sucrose density gradient centrifugation. Total ribosome content was determined by integrating the monosomal and polysomal peaks and plotted. Error bars represent means ± SD, n = 3. Numbers indicate P ‐values (unpaired Student's t ‐test). 18S and 28S rRNA levels were determined by RT–qPCR across a sucrose density gradient. Bars represent mean rRNA levels in summed monosomal and polysomal fractions ± SD, n = 3; numbers indicate P ‐values (unpaired Student's t ‐test). Total ribosome occupancy of the indicated mRNAs in eIF3k‐mAID cells exposed to DMSO or IAA for 12 h was determined by RT–qPCR of RNA across a sucrose density gradient (see ). Bars represent means ± SD, n = 3; numbers indicate P ‐values (unpaired Student's t ‐test). Triplicate RT–qPCR data across the sucrose gradient are shown below the bar graphs. Equal numbers of eIF3k‐mAID cells stably expressing ectopic RPS15A (pCDH‐RPS15A) or empty vector (pCDH) were plated and counted over a period of 6 days. Graphs represent means ± SD, n = 3. Numbers indicate P ‐values (two‐stage step‐up method of Benjamini, Krieger, and Yekutieli). 1 × 10 6 eIF3k‐mAID cells stably expressing ectopic RPS15A (pCDH‐RPS15A) or empty vector (pCDH) were injected into nude mice and tumor growth was followed for 2 weeks. Graphs represent means ± SD, n = 5–7 (see Fig ). Numbers indicate P ‐values (two‐stage step‐up method of Benjamini, Krieger, and Yekutieli). Lysate of eIF3k‐mAID cells stably expressing ectopic RPS15A (pCDH‐RPS15A) or empty vector (pCDH) were separated by sucrose density gradient centrifugation. Total ribosome content was determined by integrating and summing the monosomal and polysomal peak areas. Error bars represent means ± SD, n = 3. Numbers indicate P ‐values (unpaired Student's t ‐test). Equal numbers of eIF3k‐mAID cells stably expressing ectopic RPS4X (pCDH‐RPS4X) or empty vector (pCDH) were plated and counted over a period of 6 days. Graphs represent means ± SD, n = 3. Numbers indicate P ‐values (two‐stage step‐up method of Benjamini, Krieger, and Yekutieli). Total ribosome content of these cells was determined as described in (A). Steady‐state free ribosomal pool and translation rate as a function of eIF3k concentration (top two graphs) and the steady‐state translation rate as a function of eIF3a concentration (bottom graph). The concentration of other eIF3 subunits is kept constant at 0.5. Data information: n = number of biological replicates. Source data are available online for this figure.

Article Snippet: Rabbit Polyclonal Anti‐ RPS4X (WB 1:2,000) , Abclonal , Cat # A6730; RRID: AB_2767314.

Techniques: Gradient Centrifugation, Quantitative RT-PCR, Stable Transfection, Expressing, Plasmid Preparation, Injection, Concentration Assay

Journal: The EMBO Journal

Article Title: eIF3 mRNA selectivity profiling reveals eIF3k as a cancer‐relevant regulator of ribosome content

doi: 10.15252/embj.2022112362

Figure Lengend Snippet: 5′‐UTR sequences of RPS15A and RPS4X. 5′‐TOP element known to boost translation of ribosomal protein mRNAs (Meyuhas, ) are highlighted in gold prints, the eIF3‐binding sites mapped by Lee et al and Meyer et al are highlighted in red print. The alleles created by gene editing are shown (S15A‐eIF3KO, S4X‐eIF3KO). Note that the eIF3‐binding site in RPS4X is upstream of or overlapping with the major transcription start site. Thus, the S4X‐eIF3KO truncation we failed to generate most likely abolished the transcription of RPS4X mRNA. Parental eIF3k‐mAID cells or S15A‐eIF3KO cells (clones #45 and #361) were maintained in standard media, and cell numbers were determined at various time points. Data represent means ± SD, n = 3. Asterisks denote: * P < 0.005, ** P < 0.00005, *** P < 0.000005 (two‐stage step‐up method of Benjamini, Krieger, and Yekutieli). Relative levels of mRNAs encoding RPS15A , RPS4X , and RPL7A before and after depletion of eIF3k were determined in the indicated cell lines by RT–qPCR. Data were normalized to the signal obtained for GAPDH. Bars represent means ± SD, n = 3; numbers indicate P ‐values (unpaired Student's t ‐test). Relative translational efficiencies (TEs) of mRNAs encoding RPS15A , RPS4X , and RPL7A before and after depletion of eIF3k were determined in the indicated cell lines. RT–qPCR was performed on total RNA and on RNA contained within polysomal fractions > 2 ribosomes, and TE was calculated according to the formula TE = polysomal mRNA / total mRNA. Bars represent means ± SD, n = 3; numbers indicate P ‐values (unpaired Student's t ‐test). Basal expression of the indicated proteins was determined in parental eIF3k‐mAID cells or S15A‐eIF3KO cells (clones #45 and #361) by immunoblotting, followed by quantification of the blots. Bars represent means ± SD, n = 3 (Fig ). Numbers indicate P ‐values (unpaired Student's t ‐test). Data information: n = number of biological replicates. Source data are available online for this figure.

Article Snippet: Rabbit Polyclonal Anti‐ RPS4X (WB 1:2,000) , Abclonal , Cat # A6730; RRID: AB_2767314.

Techniques: Binding Assay, Clone Assay, Quantitative RT-PCR, Expressing, Western Blot

Journal: The EMBO Journal

Article Title: eIF3 mRNA selectivity profiling reveals eIF3k as a cancer‐relevant regulator of ribosome content

doi: 10.15252/embj.2022112362

Figure Lengend Snippet:

Article Snippet: Rabbit Polyclonal Anti‐ RPS4X (WB 1:2,000) , Abclonal , Cat # A6730; RRID: AB_2767314.

Techniques: Recombinant, Plasmid Preparation, Sequencing, Protease Inhibitor, Magnetic Beads, Software, Microscopy, Microarray, Imaging, Flow Cytometry, Mutagenesis, Gel Extraction, cDNA Synthesis, Sample Prep, Clear Native PAGE, Bicinchoninic Acid Protein Assay