rabbit antibodies against rps3  (Proteintech)

Journal: Cell

Article Title: Ribosomal protein S3: a KH domain subunit in NF-kappaB complexes that mediates selective gene regulation.

doi: 10.1016/j.cell.2007.10.009

Figure Lengend Snippet: Figure 1. The Interaction between p65 and RPS3 (A) The amino acid sequence and a diagram of RPS3; the residues detected by mass spectrometry are shown in red. NLS, nuclear local- ization signal; KH, K homology. (B) Pull-down with GST-S3 or GST proteins with nuclear extracts from TCR-stimulated Jurkat cells, followed by immunoblotting for p65 or GST. (C) Whole-cell lysates from Jurkat cells were immunoblotted directly or after immunoprecipitation with RPS3 antiserum (S3) or preimmune serum (Ig) for the indicated proteins. (D) Lysates as in (C) were immunoblotted for RPS3 directly or after immunoprecipitation with p65, p50, or isotype control (iso) antibodies.

Article Snippet: Rabbit antibodies against RPS3 were custom prepared by Proteintech Group, Inc. (Hegde et al., 2007) and by PrimmBiotech, Inc. (Cambridge, MA, USA).

Techniques: Sequencing, Mass Spectrometry, Western Blot, Immunoprecipitation, Control

Journal: Cell

Article Title: Ribosomal protein S3: a KH domain subunit in NF-kappaB complexes that mediates selective gene regulation.

doi: 10.1016/j.cell.2007.10.009

Figure Lengend Snippet: Figure 3. Independent Nuclear Translocation of p65 and RPS3 (A) Confocal micrographs of Jurkat cells stimulated with no treatment (NT), aCD3/CD28 (TCR), TNFa (TNF), or Geldanamycin (GA). The fixed cells were stained with FITC-anti-RPS3 (green), Alexa Fluor 594-anti-p65 (red), and the nuclear dye Hoechst 33342 (blue). (B) Immunoblots with the indicated antibodies of Jurkat cells that were transfected with NS, RPS3 (S3), and p65 siRNAs, TCR-stimulated 72 hr after transfection, and then fractionated into cytosolic and nuclear extracts. PARP was a nuclear fraction control. (C) Confocal micrographs of Jurkat cells treated as described above, fixed, and stained with FITC-anti-p65 (green) and Hoechst (red). Percentages of cells with nuclear p65 after TCR stimulation are quantified below. (D) Jurkat cells treated as in (C) and stained with FITC-anti-RPS3 (green) and Hoechst (red). Percentages of cells with nuclear RPS3 after TCR stim- ulation are quantified below. The scale bars are 10 mm.

Article Snippet: Rabbit antibodies against RPS3 were custom prepared by Proteintech Group, Inc. (Hegde et al., 2007) and by PrimmBiotech, Inc. (Cambridge, MA, USA).

Techniques: Translocation Assay, Staining, Western Blot, Transfection, Control

Journal: Cell

Article Title: Ribosomal protein S3: a KH domain subunit in NF-kappaB complexes that mediates selective gene regulation.

doi: 10.1016/j.cell.2007.10.009

Figure Lengend Snippet: Figure 4. RPS3 Augments NF-kB Signaling through Association with p65 (A) Jurkat cells were cotransfected as in Figure 2A with the indicated siRNA and the amount of pHA-p65 plasmid shown and, 48 hr later, were analyzed by luciferase assay. (B) Jurkat cells treated and analyzed as in (A) with the indicated amount of pcDNA-IKKb plasmid. (C) Jurkat cells were transfected with pHA-p65 (p65) plus 3FLAG-RPS3 (S3) plasmids as shown and were analyzed as in (A). (D) Diagram of the truncation mutants of p65 fused to EGFP. RHD, Rel homology domain; NTD, N-terminal domain; DimD, dimerization domain; TAD, transcriptional activation domain. (E) 293T cells transfected with constructs expressing full-length or truncated p65-GFP fusion proteins, lysed after 24 hr, and immunoblotted for GFP directly (input, right panel) or after immunoprecipitation with RPS3 antiserum (immunoprecipitation, RPS3, left panel). The blots at the bottom show RPS3 in each sample. (F) Diagram of RPS3 and FLAG-tagged truncation mutants. KH, K homology. (G) 293T cells transfected with constructs expressing full-length or truncated FLAG-tagged RPS3 proteins, lysed after 24 hr, and immunoblotted (IB) for FLAG-RPS3 or p65 after immunoprecipitation with FLAG antibody or for p65 in lysates (Input WB: p65).

Article Snippet: Rabbit antibodies against RPS3 were custom prepared by Proteintech Group, Inc. (Hegde et al., 2007) and by PrimmBiotech, Inc. (Cambridge, MA, USA).

Techniques: Plasmid Preparation, Luciferase, Transfection, Activation Assay, Construct, Expressing, Immunoprecipitation

Journal: Cell

Article Title: Ribosomal protein S3: a KH domain subunit in NF-kappaB complexes that mediates selective gene regulation.

doi: 10.1016/j.cell.2007.10.009

Figure Lengend Snippet: Figure 5. RPS3 Is Involved in NF-kB Gene Regulation (A) Jurkat cells treated with or without PMA plus ionomycin (P/I) were used for chromatin immunoprecipitation (ChIP) assays with p65 antibody (p65), RPS3 antiserum (RPS3), or control antiserum (iso). The kB sites in the IkBa promoter, IL-8 promoter, or b-actin promoter were detected by PCR. (B) Nuclear extracts of 293T cells (lanes 1–9) and 293T cells overexpressing 3FLAG-RPS3 (lanes 10–14) treated with (+) or without () TNFa were analyzed by EMSA with a 32P-labeled, double-stranded Ig kB probe; in some cases, cells were analyzed with 100-fold unlabeled wild-type (WT) Ig kB, mutant (Mut) Ig kB, or nonspecific OCT1 oligonucleotide competitors. Supershift analysis with the indicated antibodies is shown in lanes 3– 6 and 12–14. p65-p50 and p65-p65 complexes are designated, and the supershifted bands, nonspecific bands, and free oligonucleotides are labeled with dots, asterisks, and triangles, respectively. (C) Nuclear extracts of 293T cells stimulated as in (B) were analyzed by EMSA. Supershift analysis was conducted with p65, an isotype control (mIg), or different doses of purified RPS3 antibodies (lanes 19–21). The NF-kB complexes, supershifted bands, nonspecific bands, and free oligonucleotides are labeled as in (B). (D) The nuclear extracts are as in (C), and those from 70Z/3 cells stimulated with or without LPS were analyzed by EMSA and super shift analysis with 32P-labeled OCT1 (upper panel) and AP-1 (bottom panel) probes, respectively. (E) Jurkat cells transfected with NS or S3 siRNAs were stimulated with or without aCD3/CD28, and nuclear extracts were analyzed by EMSA with a 32P-labeled Ig kB probe. The labels are the same as in (C), and NF-kB complex is labeled with an arrow.

Article Snippet: Rabbit antibodies against RPS3 were custom prepared by Proteintech Group, Inc. (Hegde et al., 2007) and by PrimmBiotech, Inc. (Cambridge, MA, USA).

Techniques: Chromatin Immunoprecipitation, Control, Labeling, Mutagenesis, Transfection

Journal: Cell

Article Title: Ribosomal protein S3: a KH domain subunit in NF-kappaB complexes that mediates selective gene regulation.

doi: 10.1016/j.cell.2007.10.009

Figure Lengend Snippet: Figure 6. RPS3 Facilitates p65 Binding to kB Sites (A) Jurkat T cells silenced with nonspecific (NS) or RPS3 (S3) siRNA (left panel) or p65 siRNA (right panel) were treated as in Figure 5A for ChIP analysis with isotype (I), p65 (65), or RPS3 antibodies and PCR evaluation of the kB sites in the IL-8, IkBa, or b-actin promoters. (B) Real-time PCR quantitation of mRNA levels of IL-8 and IkBa normalized to GAPDH in Jurkat cells silenced with NS or RPS3 (S3) siRNAs and stim- ulated with no treatment (NT) or aCD3/CD28 (TCR). (C) Recombinant p65 (200 ng/sample) was incubated with increasing amounts of GST-RPS3 protein (0, 100, 200, 400, and 800 ng in lanes 1–5, respectively) or GST protein (800 ng, lane 6) at 25C for 30 min, followed by an EMSA with a 32P-labeled Ig kB probe. The p65 homodimer and free oligonucleotide were labeled by dots and a triangle, respectively. Darker and lighter autoradiographic exposures are shown. (D) The binding site occupancy of the complex shown in (C), calculated as a ratio of the intensity of the complex band to the free oligonucleotide band.

Article Snippet: Rabbit antibodies against RPS3 were custom prepared by Proteintech Group, Inc. (Hegde et al., 2007) and by PrimmBiotech, Inc. (Cambridge, MA, USA).

Techniques: Binding Assay, Real-time Polymerase Chain Reaction, Quantitation Assay, Recombinant, Incubation, Labeling

Journal: Cell

Article Title: Ribosomal protein S3: a KH domain subunit in NF-kappaB complexes that mediates selective gene regulation.

doi: 10.1016/j.cell.2007.10.009

Figure Lengend Snippet: Figure 7. RPS3 Regulates a Subset of Physiologically Important NF-kB Target Genes (A) The Venn diagram shows genes that are downregulated after p65 siRNA (red), RPS3 siRNA (blue), or both in TCR-stimulated versus untreated Jurkat T cells determined by the Lymphochip microarray. Of the 3035 genes with multiple spots available, 88 (2.9%) were decreased (R1.4-fold) in the S3 sample, and 57 (1.9%) were decreased in the p65 sample. A total of 21 (37%) were downregulated in both samples (R2 repeats in both samples), which is greater than expected by random chance alone (c2 test, p < 0.001). (B) Jurkat cells treated with or without aCD3/CD28 (TCR) were analyzed by ChIP with p65 antibody (65), RPS3 antiserum (S3), or isotype control serum (iso). PCR was used to detect the promoter kB sites for CD25, CD69, IkBa, or IL-2 and the control b-actin DNA. (C) Jurkat cells were transfected with NS or S3 siRNAs, stimulated as in Figure 6A, and ChIP was performed with isotype (I) or p65 (65) antibodies as shown. PCR was used to detect the promoter kB sites for CD25, CD69, IL-2, or IL-8. (D) Human peripheral blood lymphocytes (PBLs) were transfected with NS or RPS3 (S3) siRNAs and were stimulated with aCD3/CD28 for 36 hr. IL-2 in supernatants was measured by ELISA. (E) PBLs were transfected and stimulated as in (D) only for 12 hr, and they were analyzed by flow cytometry after cell-surface staining for CD25. The percentage of CD25-positive cells is indicated. (F) 70Z/3 cells were transduced with pNUTS (vec, red) or pNUTS-RPS3-shRNA (S3, blue) lentiviruses and were stimulated with LPS or IFN-g, or were left not treated (NT), and they were analyzed by flow cytometry with live gating on GFP+, i.e., lentivirally transduced, cells and staining for Ig k light chain. RPS3 knockdown is shown by immunoblotting, with a b-actin control (inset). The mean fluorescence intensity (MFI) in relative units of cell- surface k light-chain expression in GFP+ cells is illustrated. (G) Identical amounts of nuclear extracts (NE) prepared from 70Z/3 cells transduced as in (F) and stimulated with or without LPS were analyzed by EMSA with a 32P-labeled Ig kB probe. The NF-kB band is indicated, and a nonspecific band and free oligonucleotide are labeled with an asterisk and triangle, respectively. The lower panel shows an immunoblot for p65 in the NE samples. (H) Total RNA was isolated from 70Z/3 cells transduced as in (F) and stimulated with LPS (1 mg/ml) or cycloheximide (CHX, 20 mM) for 8 hr, or not treated (NT). The mRNA levels of k light chain were measured by quantitative real-time PCR and were normalized to levels of GAPDH; the mRNA level from the cells transfected with pNUTS alone and mock treated was set as 1.

Article Snippet: Rabbit antibodies against RPS3 were custom prepared by Proteintech Group, Inc. (Hegde et al., 2007) and by PrimmBiotech, Inc. (Cambridge, MA, USA).

Techniques: Microarray, Control, Transfection, Enzyme-linked Immunosorbent Assay, Cytometry, Staining, Transduction, shRNA, Knockdown, Western Blot, Expressing, Labeling, Isolation, Real-time Polymerase Chain Reaction