rabbit anti gilz polyclonal antibody  (Proteintech)

Journal: iScience

Article Title: TSC22D3 as an immune-related prognostic biomarker for acute myeloid leukemia

doi: 10.1016/j.isci.2023.107451

Figure Lengend Snippet:

Article Snippet: Rabbit Polyclonal anti-TSC22D3 , Signalway Antibody , Cat#39175.

Techniques: Virus, Recombinant, Staining, Reverse Transcription, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Plasmid Preparation, Apoptosis Assay, Cell Cycle Assay, Enzyme-linked Immunosorbent Assay, RNA Sequencing, Sequencing, shRNA, Software

Journal: Frontiers in immunology

Article Title: Non-canonical Glucocorticoid Receptor Transactivation of gilz by Alcohol Suppresses Cell Inflammatory Response.

doi: 10.3389/fimmu.2017.00661

Figure Lengend Snippet: Figure 1 | Mifepristone blocks dexamethasone- but not alcohol-activation of gilz. MM6 cells were exposed to alcohol (0, 25, or 50 mM) or dexamethasone (Dex, 1 µM) for 24 h in the presence or absence of mifepristone (5 µM). GILZ expression from each treatment was measured by quantitative real-time PCR. Changes in GILZ expression were expressed by fold increases as compared to the base level of GILZ in the no treatment control. The data represent at least three independent experiments. The shown P-values by Student’s t-test indicate statistically significant differences. NS denotes non-significant difference.

Article Snippet: Rabbit polyclonal anti-human GILZ antibody at 1:200 final dilution (FL-134, Santa Cruz Biotechnology) and rabbit anti-β-Actin antibody at 1:5,000 final dilution (Cell Signaling Technology, Danvers, MA, USA) were used to probe GILZ and actin. lPs stimulation and TnF-α Measurement Different genotypes of MM6 cells, as indicated in each experiment, were exposed to alcohol (0 or 50 mM) 1 h before stimulation with 100 or 1,000 ng/ml LPS from Escherichia coli 0111:B4 (Sigma-Aldrich).

Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Control

Journal: Frontiers in immunology

Article Title: Non-canonical Glucocorticoid Receptor Transactivation of gilz by Alcohol Suppresses Cell Inflammatory Response.

doi: 10.3389/fimmu.2017.00661

Figure Lengend Snippet: Figure 2 | Alcohol activates GILZ promoter via GRE cis-elements. (A) The gilz promoter was cloned into the promoter-less pGL.4.16[luc2CP/Hygro] construct, generating the pGL-Long-GILZp-Fluc plasmid. GRE3-5 sites were removed by deletion of the region (−1,560 to −1,884), resulting in the pGL-Short-GILZp-Fluc plasmid. The GRE1 and GRE2 sites were mutated, producing the pGL-Short-mutGILZp-Luc plasmid. (B) A549 cells were, respectively, transfected with one of the three resulting plasmids in combination with the reference plasmid pGL4.75-hRluc that constitutively expressed the humanized Renilla luciferase. Forty-eight hours after transfection, the cells were exposed to 0 or 50 mM alcohol or 1 µM of Dex for 24 h. Potency of the wild-type or each mutated gilz promoter to drive the reporter gene expression was determined by the activity of the Firefly luciferase that had been normalized to that of the Renilla luciferase in each sample. Asterisks denote significant differences between the comparing groups by Student’s t-test (*p < 0.05, **p < 0.01; n = 3).

Article Snippet: Rabbit polyclonal anti-human GILZ antibody at 1:200 final dilution (FL-134, Santa Cruz Biotechnology) and rabbit anti-β-Actin antibody at 1:5,000 final dilution (Cell Signaling Technology, Danvers, MA, USA) were used to probe GILZ and actin. lPs stimulation and TnF-α Measurement Different genotypes of MM6 cells, as indicated in each experiment, were exposed to alcohol (0 or 50 mM) 1 h before stimulation with 100 or 1,000 ng/ml LPS from Escherichia coli 0111:B4 (Sigma-Aldrich).

Techniques: Clone Assay, Construct, Plasmid Preparation, Transfection, Luciferase, Gene Expression, Activity Assay

Journal: Frontiers in immunology

Article Title: Non-canonical Glucocorticoid Receptor Transactivation of gilz by Alcohol Suppresses Cell Inflammatory Response.

doi: 10.3389/fimmu.2017.00661

Figure Lengend Snippet: Figure 7 | A proposed model for alcohol induction of GIlZ gene expression via a non-canonical glucocorticoid receptor (GR) transactivation. Left panel: glucocorticoids (GC) activation of GR signaling. Cortisol as a representative for GCs enters the cytoplasm and binds to GR. Such a binding alters the GR configuration and disassembles the GR complex. Then, the GC-coupled GR is translocated into the nucleus to bind GREs for activation of GR-targeting genes, such as gilz. Right panel: Alcohol activation of GR signaling. Alcohol enters the cytoplasm causing disassembly of the GR complex. The freed non-ligand-bound GR migrates to the nucleus and binds GREs for activation of GR-targeting genes, such as gilz. Heat shock proteins and p23 represent the chaperone proteins in the cytoplasmic GR complex.

Article Snippet: Rabbit polyclonal anti-human GILZ antibody at 1:200 final dilution (FL-134, Santa Cruz Biotechnology) and rabbit anti-β-Actin antibody at 1:5,000 final dilution (Cell Signaling Technology, Danvers, MA, USA) were used to probe GILZ and actin. lPs stimulation and TnF-α Measurement Different genotypes of MM6 cells, as indicated in each experiment, were exposed to alcohol (0 or 50 mM) 1 h before stimulation with 100 or 1,000 ng/ml LPS from Escherichia coli 0111:B4 (Sigma-Aldrich).

Techniques: Gene Expression, Activation Assay, Binding Assay

Journal: PloS one

Article Title: Yersinia enterocolitica YopT and Clostridium difficile toxin B induce expression of GILZ in epithelial cells.

doi: 10.1371/journal.pone.0040730

Figure Lengend Snippet: Figure 1. Yersinia enterocolitica induces GILZ expression. HeLa cells were infected with a strain harboring the Yersinia virulence plasmid (pYV+) or plasmid cured strain (pYV2) with MOI 20 or stimulated with 100 mM DEX for indicated time intervals. The amount of GILZ in cytosolic proteins lysates of HeLa cells was detected by immunoblot at different time points. Actin was used as an internal standard. A representative experiment and quantification means + SEM normalized to untreated of at least 3 experiments are shown. doi:10.1371/journal.pone.0040730.g001

Article Snippet: For detection of GILZ expression the polyclonal rabbit anti-GILZ antibody was used followed by incubation with a goat anti-rabbit IgG (H+L) as a secondary antibody and a horseradish peroxidase-conjugated (HRP) donkey anti-goat IgG (H+L) antibody (Jackson Immuno Research Laboratories, West Grove, PA) as a tertiary antibody.

Techniques: Expressing, Infection, Plasmid Preparation, Western Blot

Journal: PloS one

Article Title: Yersinia enterocolitica YopT and Clostridium difficile toxin B induce expression of GILZ in epithelial cells.

doi: 10.1371/journal.pone.0040730

Figure Lengend Snippet: Figure 2. Yersinia induced GILZ expression depends on YopT protease activity. HeLa cells were infected with (A) strains with (pYV+) or without (pYV2) pathogenicity plasmid or with a pathogenicity plasmid derivate coding for a functional translocation apparatus but not for effector Yops (pTTSS) or with a pathogenicity plasmid coding for a defective translocation apparatus (pYV5.15) with MOI 20 for 2 or 4 h to determine GILZ mRNA expression by real-time RT-PCR. Means + SD of 2 independent experiments. Significant differences compared to pYV+ are indicated by asterisks (p,0.05). (B) Immunoblot of GILZ protein expression induced by strains with single yop deletions. Representative immunoblot and quantification means + SEM of 4 independent experiments normalized to untreated. Significant differences compared to DyopT are indicated by asterisks (p,0.05). (C) Infection with a yopT deletion strain and derivative strains complemented with an additional plasmid encoding wildtype (pyopT) or protease deficient (pyopTC139A) YopT. Representative immunoblot analysis of GILZ protein expression in the cytosolic fraction and of RhoA in cytosolic and membrane fraction (left) and quantification means + SD of GILZ expression of two representative experiments normalized to untreated (right).

Article Snippet: For detection of GILZ expression the polyclonal rabbit anti-GILZ antibody was used followed by incubation with a goat anti-rabbit IgG (H+L) as a secondary antibody and a horseradish peroxidase-conjugated (HRP) donkey anti-goat IgG (H+L) antibody (Jackson Immuno Research Laboratories, West Grove, PA) as a tertiary antibody.

Techniques: Expressing, Activity Assay, Infection, Plasmid Preparation, Functional Assay, Translocation Assay, Quantitative RT-PCR, Western Blot, Membrane

Journal: PloS one

Article Title: Yersinia enterocolitica YopT and Clostridium difficile toxin B induce expression of GILZ in epithelial cells.

doi: 10.1371/journal.pone.0040730

Figure Lengend Snippet: Figure 3. GILZ is expressed upon stimulation with C3 exotoxin or toxin B. (A) HeLa cells were incubated with toxin B (50ng/ml) for indicated the time spans and immunoblots were performed from whole cell lysates using anti-Rac1 mAb102 recognizing only non-glucosylated Rac-1, and an anti-Rac1 mAb antibody recognizing total Rac1 as well as antibodies recognizing RhoB, actin or GILZ. (B) Immunoblot of GILZ and actin expression upon stimulation of HeLa cells with C2IN-C3lim (100 ng/mL) + C2IIa (200 ng/mL) for indicated time spans. (C) To explore the expression of additional GILZ isoforms, HeLa cells were transfected with 7.5 nM siRNA specific for GILZ or control siRNA for 48 h and subsequently either left untreated or stimulated with C. difficile toxin B (50 ng/ml) or 100 mM DEX for 4 h. Arrows mark three GILZ isoforms which were inhibited by the used GILZ siRNA. Note that only isoform 1 was induced by the used stimuli. (D) Cells were stimulated with toxin B for 2 or 4 h to determine GILZ mRNA expression by real-time RT-PCR. Mean + SD of 2 independent experiments normalized to untreated. (E) To assay transcriptional activity of the GILZ promoter cells were transfected with a luciferase reporter under control of a 2088 bp GILZ promoter and co-transfected with pCMV-ß-gal (for standardization) 24 h before infection with a Y. enterocolitica pYV+ and various mutant strains or treatment with DEX or toxin B. Means + SD of 4 independent experiments normalized to untreated. Significant differences compared to untreated are indicated by asterisks (p,0.05). doi:10.1371/journal.pone.0040730.g003

Article Snippet: For detection of GILZ expression the polyclonal rabbit anti-GILZ antibody was used followed by incubation with a goat anti-rabbit IgG (H+L) as a secondary antibody and a horseradish peroxidase-conjugated (HRP) donkey anti-goat IgG (H+L) antibody (Jackson Immuno Research Laboratories, West Grove, PA) as a tertiary antibody.

Techniques: Incubation, Western Blot, Expressing, Transfection, Control, Quantitative RT-PCR, Activity Assay, Luciferase, Infection, Mutagenesis

Journal: PloS one

Article Title: Yersinia enterocolitica YopT and Clostridium difficile toxin B induce expression of GILZ in epithelial cells.

doi: 10.1371/journal.pone.0040730

Figure Lengend Snippet: Figure 4. Role of Rho GTPases and MAP kinases for GILZ expression. (A) Overexpression of RhoA or RhoB lowers basal GILZ levels. HeLa cells were co-transfected with the p2088 GILZ promoter luciferase reporter and pHM6 based plasmid for overexpression of the indicated Rho GTPases. Means + SD of 3 independent experiments normalized to untreated. Significant differences compared to control vector transfection are indicated by asterisks (p,0.05). In a control experiment, HeLa cells were transfected in the same setting and cell lysates were used for immunoblots to detect RhoA, RhoB, Cdc42 and Rac1 expression. (B) HeLa cells were transfected for 48 h with indicated concentrations of siRNA. Immunoblots were performed from cell lysates for RhoA and RhoB and from cytosolic extracts for GILZ. (C) Toxin B treatment leads to fast and transient MAPK phosphorylation. After treatment of HeLa cells with toxin B for the indicated time spans, levels of phosphorylated as well as total ERK and p38 were assayed by immunoblot. (C) Toxin B induced GILZ expression is mediated by both ERK and p38 MAPK. Cells were pretreated with MAPK phosphorylation inhibitors SB 202190 (p38) or PD 98059 (ERK) 2 h prior to toxin B stimulation and GILZ protein was detected by immunoblot analysis at 6 h or 24 h after stimulation. doi:10.1371/journal.pone.0040730.g004

Article Snippet: For detection of GILZ expression the polyclonal rabbit anti-GILZ antibody was used followed by incubation with a goat anti-rabbit IgG (H+L) as a secondary antibody and a horseradish peroxidase-conjugated (HRP) donkey anti-goat IgG (H+L) antibody (Jackson Immuno Research Laboratories, West Grove, PA) as a tertiary antibody.

Techniques: Expressing, Over Expression, Transfection, Luciferase, Plasmid Preparation, Control, Western Blot, Phospho-proteomics

Journal: PloS one

Article Title: Yersinia enterocolitica YopT and Clostridium difficile toxin B induce expression of GILZ in epithelial cells.

doi: 10.1371/journal.pone.0040730

Figure Lengend Snippet: Figure 5. Differential activation of truncated GILZ promoter elements by toxin B or DEX. HeLa cells were transiently transfected with pCMV-ß-gal for 24 h and co-transfected (A) with p2088-Luc or luciferase reporters fused to shortened promoter regions, containing the indicated TF binding sites. GRE: glucocorticoid responsive element, FHRE: forkhead responsive elements, c-myc: c-myc binding site (E-box), Oct: octamer binding site (B) Transfected cells were treated with 100 mM DEX or 50 ng/ml toxin B for 6 h. Subsequently luciferase assays were performed. Results are expressed as fold induction compared to unstimulated (none) cells and represent the mean + SEM of three experiments performed in triplicates (p,0.05). doi:10.1371/journal.pone.0040730.g005

Article Snippet: For detection of GILZ expression the polyclonal rabbit anti-GILZ antibody was used followed by incubation with a goat anti-rabbit IgG (H+L) as a secondary antibody and a horseradish peroxidase-conjugated (HRP) donkey anti-goat IgG (H+L) antibody (Jackson Immuno Research Laboratories, West Grove, PA) as a tertiary antibody.

Techniques: Activation Assay, Transfection, Luciferase, Binding Assay

Journal: PloS one

Article Title: Yersinia enterocolitica YopT and Clostridium difficile toxin B induce expression of GILZ in epithelial cells.

doi: 10.1371/journal.pone.0040730

Figure Lengend Snippet: Figure 6. Importance of specific cis-elements for toxin B induced GILZ promoter trans-activation. (A) Recognition sequences of cis- elements which were mutated. Mutated base pairs are highlighted using bold letters. (B) HeLa cells were transfected with p1940-Luc and different mutated derivatives for 24 h and subsequently stimulated with DEX or toxin B for 6 h. Data are shown as relative light units (RLU) standardized to b- Gal activity and protein concentration or (C) as fold induction after DEX or toxin B stimulation compared to untreated conditions of each individual expression vector. Means + SEM of four independent experiments are shown. Asterisks indicate significant differences between DEX or toxin B stimulation compared to uninfected (p,0.05). doi:10.1371/journal.pone.0040730.g006

Article Snippet: For detection of GILZ expression the polyclonal rabbit anti-GILZ antibody was used followed by incubation with a goat anti-rabbit IgG (H+L) as a secondary antibody and a horseradish peroxidase-conjugated (HRP) donkey anti-goat IgG (H+L) antibody (Jackson Immuno Research Laboratories, West Grove, PA) as a tertiary antibody.

Techniques: Activation Assay, Transfection, Activity Assay, Protein Concentration, Expressing, Plasmid Preparation

Journal: PloS one

Article Title: Yersinia enterocolitica YopT and Clostridium difficile toxin B induce expression of GILZ in epithelial cells.

doi: 10.1371/journal.pone.0040730

Figure Lengend Snippet: Figure 7. Role of myc-1 E-box in TF binding. Electromobility shift analyses were performed using a double-stranded oligonucleotide probe representing the GILZ promoter sequence 263 to 237 and for some experiments probes with mutations of the E-box cis-elements and the flanking cAMP response (Cre) elements as depicted in (A). HeLa cells were stimulated/infected for 0.5 h or indicated time intervals with toxin B (B, D, E) or Y. enterocolitica (C) and nuclear extracts of these cells were incubated with P32-labeled GILZ263/237 probe. Subsequently band shift analyses were performed. (D) Nuclear extracts were pretreated with a 100-fold excess of indicated cold probes. (E) Nuclear extracts were pretreated with indicated antibodies. Anti-p65 antibody was used as a negative control. doi:10.1371/journal.pone.0040730.g007

Article Snippet: For detection of GILZ expression the polyclonal rabbit anti-GILZ antibody was used followed by incubation with a goat anti-rabbit IgG (H+L) as a secondary antibody and a horseradish peroxidase-conjugated (HRP) donkey anti-goat IgG (H+L) antibody (Jackson Immuno Research Laboratories, West Grove, PA) as a tertiary antibody.

Techniques: Binding Assay, Sequencing, Infection, Incubation, Labeling, Electrophoretic Mobility Shift Assay, Negative Control

Journal: PloS one

Article Title: Yersinia enterocolitica YopT and Clostridium difficile toxin B induce expression of GILZ in epithelial cells.

doi: 10.1371/journal.pone.0040730

Figure Lengend Snippet: Figure 8. Role of USF-1 and USF-2 for toxin B induced GILZ expression. (A) HeLa cells were transfected with siRNA silencing USF-1 or USF-2 or with control siRNA (siC) 48 h prior to toxin B treatment and USF-1/2 as wells as GILZ and actin expression was determined by immunoblot. (B) HeLa cells were transfected with empty vector or a dominant negative (DN) USF-1 mutant and subsequently GILZ mRNA or GILZ protein expression was determined by real time RT-PCR (one representative experiment performed in quadruplicates, means + SEM) or immunoblot. doi:10.1371/journal.pone.0040730.g008

Article Snippet: For detection of GILZ expression the polyclonal rabbit anti-GILZ antibody was used followed by incubation with a goat anti-rabbit IgG (H+L) as a secondary antibody and a horseradish peroxidase-conjugated (HRP) donkey anti-goat IgG (H+L) antibody (Jackson Immuno Research Laboratories, West Grove, PA) as a tertiary antibody.

Techniques: Expressing, Transfection, Control, Western Blot, Plasmid Preparation, Dominant Negative Mutation, Mutagenesis, Quantitative RT-PCR