Journal: Scientific Reports

Article Title: Tumor suppression via inhibition of SWI/SNF complex-dependent NF-κB activation

doi: 10.1038/s41598-017-11806-9

Figure Lengend Snippet: Microarray analysis of NF-κB target genes induced by TNF-α in A549 and HeLaS3 cells. ( a ) Heat map generated by microarray analysis of reported NF-κB target genes. Cells transduced with Halo-DPF3-CT1–expressing retrovirus vector or empty vector (EV-2) were grown in the absence (TNF-α: −) or presence (TNF-α: +) of TNF-α (10 µg/ml) for 1 h. Total RNA was isolated and analyzed by microarray as described in the Materials and Methods. Quantile normalized expression data were calculated using R package. All reported NF-κB target genes (Boston University; http://www.bu.edu/nf-kb/ ) were clustered using four RNA samples (EV-1 ± TNF-α and Halo-DPF3-CT1 ± TNF-α). Bars on the right indicate the gene cluster regions including TNF-α inducible genes in these cells. ( b , c ) Heat map of TNF-α–induced NF-κB target genes that were either CT1-sensitive or CT1-insensitive. From the list of NF-κB target genes, we selected TNF-α inducible genes in A549 or HeLaS3 cells using the following criteria: expression ratio of TNF-α–treated EV-1 transduced cells to untreated EV-1 transduced cells >1.5 and a Z-score > 2. From these TNF-α inducible genes in either A549 or HeLaS3 cells (a total of 71 genes), CT1-sensitive genes ( b ) were further selected by the following criteria: ratio of CT1-expressing cells to EV-2 transduced cells <0.66 and a Z-score < −2. The other genes were classified as CT1-insensitive ( c ).

Article Snippet: The human cancer cell lines A549 (non-small-cell lung carcinoma; American Type Culture Collection), H1299 (non-small-cell lung carcinoma; American Type Culture Collection), HeLaS3 (cervical carcinoma; Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, Japan), and MDA-MB-231 (human breast cancer; American Type Culture Collection) were used in the present study.

Techniques: Microarray, Generated, Transduction, Expressing, Plasmid Preparation, Isolation

Journal: Scientific Reports

Article Title: Tumor suppression via inhibition of SWI/SNF complex-dependent NF-κB activation

doi: 10.1038/s41598-017-11806-9

Figure Lengend Snippet: Suppression of TNF-α inducible NF-κB target genes by CT1. ( a , b ) A549 or HeLaS3 cells were transduced with Halo-DPF3-CT1 expressing retrovirus vector or empty vector (EV-2). The cells were then stimulated with TNF-α for 1 h ( a ) or for 30, 60, 90, and 120 min ( b ) and total RNA was prepared. The mRNA levels of IL6 , IL8 , TNF , and ICAM1 were determined by qRT-PCR. The ratios of these mRNA levels to those of A549 (or HeLaS3) cells that were transduced with EV-2 (control) are shown in ( a ). In ( b ), RNA levels before the TNF-α treatment were taken as 1.0. ( c ) RNA was extracted from parallel cultures that were maintained in the absence of TNF-α treatment, and the mRNA levels of these four genes were determined and presented as in ( a ). ( d ) Scatter diagram comparing the effect of CT1 suppression on the basal expression (CT1–/EV-2−) and TNF-α–induced expression (CT1+/EV-2+) of all NF-κB target genes that were induced by TNF-α in either A549 or HeLaS3 cells. The NF-κB target genes listed in Supplementary Table were plotted (ratio of EV-2(+) to EV-2(−) > 1.5, Z-score > 2.0). White boxes: CT1-sensitive genes in HeLaS3, Grey boxes: CT1-insensitive genes in HeLaS3. White circles: CT1-sensitive genes in A549, Grey circles: CT1-insensitive genes in A549. ( a – c ), The data present means ± SD ( n = 3).

Article Snippet: The human cancer cell lines A549 (non-small-cell lung carcinoma; American Type Culture Collection), H1299 (non-small-cell lung carcinoma; American Type Culture Collection), HeLaS3 (cervical carcinoma; Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, Japan), and MDA-MB-231 (human breast cancer; American Type Culture Collection) were used in the present study.

Techniques: Transduction, Expressing, Plasmid Preparation, Quantitative RT-PCR, Control

Journal: Scientific Reports

Article Title: Tumor suppression via inhibition of SWI/SNF complex-dependent NF-κB activation

doi: 10.1038/s41598-017-11806-9

Figure Lengend Snippet: Effects of IL6 knockdown or the addition of IL-6 protein on cellular growth. ( a , b ) Effects of shIL6 expression on the growth of monolayer cultures ( a ) and soft agar cultures ( b ) examined by the same procedure described for Fig. . A549 or HeLaS3 cells were transduced with shRNA expressing vectors. The knockdown efficiency of shIL6#1 was calculated as 0.28 ± 0.014 in MDA-MB-231 cells. Colony numbers formed by cells transduced with shCre#4 (negative control) were assigned a value of 1.0. ( c ) The suppression of anchorage-independent growth by the exogenous expression of HA-DPF3-CT1 can be rescued by IL-6. A549 cells transduced with HA-DPF3-CT1 were kept in soft agar in the absence or presence of IL-6 (10 ng/ml). Colony numbers formed by cells transduced with EV-3 were assigned a value of 1.0. ( a , b , c ) The data present means ± SD ( n = 3). *P < 0.05, ***P < 0.001.

Article Snippet: The human cancer cell lines A549 (non-small-cell lung carcinoma; American Type Culture Collection), H1299 (non-small-cell lung carcinoma; American Type Culture Collection), HeLaS3 (cervical carcinoma; Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, Japan), and MDA-MB-231 (human breast cancer; American Type Culture Collection) were used in the present study.

Techniques: Knockdown, Expressing, Transduction, shRNA, Negative Control

Journal: Cancer Cell

Article Title: Human Tumor-Associated Macrophage and Monocyte Transcriptional Landscapes Reveal Cancer-Specific Reprogramming, Biomarkers, and Therapeutic Targets

doi: 10.1016/j.ccell.2019.02.009

Figure Lengend Snippet:

Article Snippet: Migration was recorded every hour for 72 hr using the IncuCyte system (Essen Bioscience) and number of cells migrated was calculated using IncuCyte quantification software.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Chemotaxis Assay, Microarray, Software

Journal: Immunity

Article Title: Anti-commensal IgG Drives Intestinal Inflammation and Type 17 Immunity in Ulcerative Colitis

doi: 10.1016/j.immuni.2019.02.006

Figure Lengend Snippet:

Article Snippet: Human primers: CXCL8 (Hs00174103_m1), FCER1G (Hs00175408_m1), FCGR1B (Hs02341825_m1), FCGR2A (Hs01013401_g1), FCGR2B (Hs01634996_s1), FCGR3A (Hs02388314_m1), FCGR3B (Hs04334165_m1), GAPDH (Hs02786624_g1), HPRT1 (Hs02800695_m1), and IL1B (Hs01555410_m1). qPCR was carried performed on the Viia 7 PCR machine (Life Technologies).

Techniques: Virus, Control, Recombinant, Staining, SYBR Green Assay, Protease Inhibitor, Protein Extraction, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Chromatography, Library Quantification, SNP Genotyping Assay, Gene Expression, Microarray, Infection, Software, Transfection, Transferring

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: Anti-SEMA3A Antibody: A Novel Therapeutic Agent to Suppress Glioblastoma Tumor Growth

doi: 10.4143/crt.2017.315

Figure Lengend Snippet: SEMA3A is highly expressed in glioblastoma (GBM) specimens compared to the normal brains. (A) REMBRANDT microarray data analysis for SEMA3A mRNA expression levels corresponding to glioma grades II-IV. WHO, World Health Organization. *** p < 0.001. (B) Kaplan-Meier survival curves of GBM patient corresponding to SEMA3A expression levels in The Cancer Genome Atlas datasets. (C) Immunohistochemistry of SEMA3A on tissue microarray. (D) Representative analysis of SEMA3A expressions in 27 GBM paired tumor and normal specimens.

Article Snippet: The antigens that were used for panning were recombinant human SEMA3A (rhSEMA3A)/Fc chimeric protein (R&D Systems, Minneapolis, MN) and Erbitux (Eli Lilly, Indianapolis, IN).

Techniques: Microarray, Expressing, Immunohistochemistry

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: Anti-SEMA3A Antibody: A Novel Therapeutic Agent to Suppress Glioblastoma Tumor Growth

doi: 10.4143/crt.2017.315

Figure Lengend Snippet: Identification of SEMA3A-specific single chain fragment variant (scFv) using synthetic phage library. (A) The efficacy of classical panning was evaluated by the ratio of output phage titer to input phage titer. (B) Based on enzyme-linked immunosorbent assay (ELISA) results, 52 positive clones specific to human SEMA3A were selected from among 86 clones which were selected randomly in the fourth round of the output mixture. (C) The three scFvs purified using Ni-NTA resin and poly-column were eluted by phosphate buffered saline with 200 mM imidazole (pH7.4). Molecular sizes of the three anti-SEMA3A clones (A08, C10, and F11) were verified by Coomassie blue staining. (D) hSEMA3A binding activities of three anti-SEMA antibodies and 12B (a negative control) were confirmed by indirect ELISA. 96-well plates coated with hSEMA3A (1 μg/mL) or bovine serum albumin (BSA; 1 μg/mL) were bound for three anti-SEMA3A antibodies and 12B. Then, antihemaglutinin antibody conjugated to horseradish peroxidase (HRP) was used for bound scFvs detection. The absorbance of each well for HRP reactions was measured at 450 nm. OD, optical density.

Article Snippet: The antigens that were used for panning were recombinant human SEMA3A (rhSEMA3A)/Fc chimeric protein (R&D Systems, Minneapolis, MN) and Erbitux (Eli Lilly, Indianapolis, IN).

Techniques: Variant Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Purification, Saline, Staining, Binding Assay, Negative Control, Indirect ELISA

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: Anti-SEMA3A Antibody: A Novel Therapeutic Agent to Suppress Glioblastoma Tumor Growth

doi: 10.4143/crt.2017.315

Figure Lengend Snippet: Production of anti-SEMA3A IgG antibodies. (A) Purity of three anti-SEMA3A IgGs determined by high performance liquid chromatography. The peak at 15-16 minutes indicates γ-globulin (~150 kDa). The purity of all three anti-SEMA3A IgGs was over 98%. (B) Sizes of the anti-SEMA3A IgGs were verified in non-reducing (NR) and reducing (R) conditions by sodium dodecyl sulfate polyacrylamide gel electrophoresis. In NR condition, fully IgG was detected whereas in R condition where cleavages disulfide bond heavy chain and light chain were observed. (C) To determinate binding activity of human and mouse SEMA3A, three anti-SEMA3A IgGs were analyzed on enzyme-linked immunosorbent assay (ELISA) plates when the presence of hSEMA3A, mSEMA3A, and bovine serum albumin (BSA) as negative control. Optical density (OD) values derived from indirect ELISA in which SEMA3A IgG was captured via anti-human Fab antibody conjugated to horseradish peroxidase. (D) Use of the BIAcore to determine the calculated K D value of three anti-SEMA3A IgGs to human and mouse SEMA3A. The assay method was Fab-based capture format via human Fab capture kit (GE Healthcare).

Article Snippet: The antigens that were used for panning were recombinant human SEMA3A (rhSEMA3A)/Fc chimeric protein (R&D Systems, Minneapolis, MN) and Erbitux (Eli Lilly, Indianapolis, IN).

Techniques: High Performance Liquid Chromatography, Polyacrylamide Gel Electrophoresis, Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Negative Control, Derivative Assay, Indirect ELISA

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: Anti-SEMA3A Antibody: A Novel Therapeutic Agent to Suppress Glioblastoma Tumor Growth

doi: 10.4143/crt.2017.315

Figure Lengend Snippet: Anti-SEMA3A IgG antibody impedes migration and proliferation of patient-derived glioblastoma (GBM) cells. (A) The concentration of SEMA3A secreted for a day was measured by sandwich enzyme-linked immunosorbent assay (ELISA) in various cell types. GBM patient cells and U87-MG cells as a positive control and neural progenitor cell and medium as negative controls. (B) Neutralization assay of three anti-SEMA3A IgGs in U87-MG cells. The evaluate method is same method for U87-MG cells were incubated with anti-SEMA3A IgG and the amount of SEMA3A in medium was measured by sandwich ELISA. (C) While the migration rate of cells (U87-MG, 131, and 83) treated with control human IgG remains unchanged, migration rates of cells treated with anti-SEMA3A IgGs for 1 day were impeded significantly. Each antibody treatment concentration was 10 μg/mL and macroscopic observation of the transwell chambers. *p < 0.05, **p < 0.01. (D) Inhibition of migration activity by anti-SEMA3A F11 for one day was evaluated using Oris cell migration assay. The level of migration activity inhibition was proportional to the concentration gradient of anti-SEMA3A F11. Migration activity was reduced by 52% compared to the control IgG-treated cells in the max concentration of F11 (200 nM) treated cells. (E) Comparison of the effect of anti-SEMA3A F11 and control human IgG (2 μM) on in-vitro proliferation of 131 cells. On the 9 days after F11 treatment, 131 proliferation was inhibited by 60% of control human IgG treated cells. (F) Immunoblots of phospho-ERK and ERK in control human IgG or F11 IgG treated 131 and 559 cells. The SEMA3A hypersecreting 131 cells decreased ERK phosphorylation during F11 treatment and remained unchanged for ERK phosphorylation of 559 cells that secretes less SEMA3A.

Article Snippet: The antigens that were used for panning were recombinant human SEMA3A (rhSEMA3A)/Fc chimeric protein (R&D Systems, Minneapolis, MN) and Erbitux (Eli Lilly, Indianapolis, IN).

Techniques: Migration, Derivative Assay, Concentration Assay, Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Positive Control, Neutralization, Incubation, Control, Inhibition, Activity Assay, Cell Migration Assay, Comparison, In Vitro, Western Blot, Phospho-proteomics

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: Anti-SEMA3A Antibody: A Novel Therapeutic Agent to Suppress Glioblastoma Tumor Growth

doi: 10.4143/crt.2017.315

Figure Lengend Snippet: In vivo effects of anti-SEMA3A antibody on the glioblastoma (GBM) tumor growth. (A) Antitumor activity of antiSEMA3A F11 in GBM patient cell 131 xenograft model. When tumors were approximately 80 mm 3 , the patient-derived xenograft tumors were randomly assigned into study groups (n=7 mice/group) and were participated in the dosing phase of the study. Treatment of F11 was administered intravenously at 5 mg/kg and 25 mg/kg with all treatments well tolerated and anti-SEMA3A F11 injections were stopped at day 28. *p < 0.05. (B) Mass weight variation of mice was similar to mass size affected by anti-SEMA3A F11 within groups. *p < 0.05. (C, D) Representative immunofluorescence images of SEMA3A, p-ERK, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and Iba1 staining using paraffin sections of tumors. Scale bars=20 μm (white), 100 μm (red).

Article Snippet: The antigens that were used for panning were recombinant human SEMA3A (rhSEMA3A)/Fc chimeric protein (R&D Systems, Minneapolis, MN) and Erbitux (Eli Lilly, Indianapolis, IN).

Techniques: In Vivo, Activity Assay, Derivative Assay, Immunofluorescence, TUNEL Assay, Staining

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