Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Helicobacter Pylori Induces GATA3-Dependent Chitinase 3 Like 1 (CHI3L1) Upregulation and Contributes to Vascular Endothelial Injuries

doi: 10.12659/MSM.916311

Figure Lengend Snippet: The expression of GATA3 and CHI3L1 were upregulated in HUVECs co-cultured with Helicobacter pylori . ( A ) qRT-PCR was used to show the expression of GATA3 and CHI3LI mRNA in HUVECs after being cultured with medium added with CagA − H. pylori or CagA + H. pylori or PBS for 48 hours. ( B, C ) Western blot was used to show the elevated expression of GATA3 and CHI3L1 protein of the aforementioned cells, Calnexin levels were used as a loading control. * P <0.05, ** P <0.01, *** P <0.0001 **** P <0.0001 versus NC group. # P <0.05, ## P <0.01 versus CagA − group. HUVECs – human umbilical endothelial cells; CagA – cytotoxin-related protein; qRT-PCR – quantitative real-time polymerase chain reaction; PBS – phosphate-buffered saline.

Article Snippet: They were then microwaved in 0.1 M citrate solution high temperature antigen retrieval (pH 6.0) for 10 minutes; after sectioning, slices were incubated with 3% H 2 O 2 for 20 minutes at room temperature, before being incubated with goat serum for 20 minutes at room temperature and then incubated with anti-GATA3 rabbit polyclonal antibody (1: 200, Cusabio, China) and anti-CHI3L1 mouse polyclonal antibody (1: 100, Cusabio, China) at 4°C under humid conditions overnight.

Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Western Blot, Control, Real-time Polymerase Chain Reaction, Saline

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Helicobacter Pylori Induces GATA3-Dependent Chitinase 3 Like 1 (CHI3L1) Upregulation and Contributes to Vascular Endothelial Injuries

doi: 10.12659/MSM.916311

Figure Lengend Snippet: The expression of GATA3 and CHI3L1 were elevated in the Helicobacter pylori infected mice model. ( A, B ) Immunohistochemical staining was used to demonstrate the upregulation of GATA3 and CHI3L1 in mice thoracic aorta after being gavaged with CagA + H. pylori or CagA − H. pylori . ( C, D ) Western blot used to show the expression of GATA3 and CHI3L1 protein of the aforementioned mice thoracic aorta, β-asctin levels were used as a loading control. * P <0.05, ** P <0.01, *** P <0.0001 **** P <0.0001 versus NC group. # P <0.05 versus CagA − group. CagA – cytotoxin-related protein.

Article Snippet: They were then microwaved in 0.1 M citrate solution high temperature antigen retrieval (pH 6.0) for 10 minutes; after sectioning, slices were incubated with 3% H 2 O 2 for 20 minutes at room temperature, before being incubated with goat serum for 20 minutes at room temperature and then incubated with anti-GATA3 rabbit polyclonal antibody (1: 200, Cusabio, China) and anti-CHI3L1 mouse polyclonal antibody (1: 100, Cusabio, China) at 4°C under humid conditions overnight.

Techniques: Expressing, Infection, Immunohistochemical staining, Staining, Western Blot, Control

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Helicobacter Pylori Induces GATA3-Dependent Chitinase 3 Like 1 (CHI3L1) Upregulation and Contributes to Vascular Endothelial Injuries

doi: 10.12659/MSM.916311

Figure Lengend Snippet: Small interfering RNA (siRNA)-mediated knockdown of GATA3 lowered Helicobacter pylori -induced expression of CHI3L1 and p38 phosphorylation ( A ) Western blot shows the expression of GATA3 and CHI3L1 protein in HUVECs after transfected with siRNA-control or siRNA-GATA3 for 24 hours and cultured with medium added with CagA − H. pylori or CagA + H. pylori or PBS for 48 hours. Calnexin levels were used as a loading control. ( B ) The expression of GATA3 was significant reduced by siRNA-GATA3 transfected. ( C ) The elevated expression of CHI3L1 caused by H. pylori was relatively decreased as GATA3 was knocked down. ( D ) The H. pylori -induced activation of p38 MAPK was attenuated in GATA3 knocked-down cells. * P <0.05, ** P <0.01, *** P <0.0001 versus si-control group. HUVECs – human umbilical endothelial cells; CagA – cytotoxin-related protein; PBS – phosphate-buffered saline MAPK – mitogen-activated protein kinase.

Article Snippet: They were then microwaved in 0.1 M citrate solution high temperature antigen retrieval (pH 6.0) for 10 minutes; after sectioning, slices were incubated with 3% H 2 O 2 for 20 minutes at room temperature, before being incubated with goat serum for 20 minutes at room temperature and then incubated with anti-GATA3 rabbit polyclonal antibody (1: 200, Cusabio, China) and anti-CHI3L1 mouse polyclonal antibody (1: 100, Cusabio, China) at 4°C under humid conditions overnight.

Techniques: Small Interfering RNA, Knockdown, Expressing, Phospho-proteomics, Western Blot, Transfection, Control, Cell Culture, Activation Assay, Saline

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Helicobacter Pylori Induces GATA3-Dependent Chitinase 3 Like 1 (CHI3L1) Upregulation and Contributes to Vascular Endothelial Injuries

doi: 10.12659/MSM.916311

Figure Lengend Snippet: Knockdown of GATA3 restored the CagA + Helicobacter pylori -induced inhibitory function in HUVECs. ( A ) The EdU staining showed the inhibited proliferation by CagA + H. pylori was restored after GATA3 knockdown. ( B ) Transwell experiments demonstrated the migration function was increased after GATA3 knockdown. ( C ) Tube formation ability was partly repaired after GATA3 knockdown. ** P <0.01 versus si-control+CagA + H group. CagA – cytotoxin-related protein; HUVECs – human umbilical endothelial cells; EdU – 5-ethynyl-2′-deoxyuridine.

Article Snippet: They were then microwaved in 0.1 M citrate solution high temperature antigen retrieval (pH 6.0) for 10 minutes; after sectioning, slices were incubated with 3% H 2 O 2 for 20 minutes at room temperature, before being incubated with goat serum for 20 minutes at room temperature and then incubated with anti-GATA3 rabbit polyclonal antibody (1: 200, Cusabio, China) and anti-CHI3L1 mouse polyclonal antibody (1: 100, Cusabio, China) at 4°C under humid conditions overnight.

Techniques: Knockdown, Staining, Migration, Control

Journal: Molecular and Cellular Biology

Article Title: GATA3 Abundance Is a Critical Determinant of T Cell Receptor β Allelic Exclusion

doi: 10.1128/MCB.00052-17

Figure Lengend Snippet: Forced expression of GATA3 in TgCd2-GATA3 mice. (A) Western blot analysis of 10 μg or 5 μg of protein recovered from total thymocytes of a TgCd2-GATA3 or wild-type (Gata3+/+) mouse. The migration position of GATA3 (48 kDa) is indicated. Lamin B was used as the normalization control. A representative blot is shown; this experiment was repeated in two independent biological replicates with two mice of each genotype; results are presented graphically on the right. (B) Quantification of GATA3 mRNA levels in DN3a (Lin− cKitlow CD25+ CD27low FSClow), DN3b (Lin− cKitlow CD25+ CD27hi FSChi), and DN4 (Lin− cKit− CD25−) stage thymocytes isolated from TgCd2-GATA3 mice or control wild-type mice by qRT-PCR. (C) Quantification of the amount of GATA3 protein by flow cytometry using the MFI (with the background intensity of IgG staining subtracted) in staged thymocytes isolated from TgCd2-GATA3 mice or control wild-type mice. (D) Absolute numbers of thymocytes in mice of each genotype according to developmental stage. Each circle represents results for an individual animal. Solid bars indicate the averages for each genotype. *, P < 0.05; NS, not significant (P > 0.05).

Article Snippet: Totals of 10 μg and 5 μg of nuclear extracts were electrophoresed on a 4-to-15% gradient precast SDS-polyacrylamide gel (Bio-Rad), transferred to a nitrocellulose membrane (Li-Cor), and first blotted by using a rabbit anti-GATA3 antibody (clone 11599 [ 32 ]).

Techniques: Expressing, Western Blot, Migration, Isolation, Quantitative RT-PCR, Flow Cytometry, Staining

Journal: Molecular and Cellular Biology

Article Title: GATA3 Abundance Is a Critical Determinant of T Cell Receptor β Allelic Exclusion

doi: 10.1128/MCB.00052-17

Figure Lengend Snippet: RNA-seq analysis of transcript abundance and V-region utilization in TgCd2-GATA3 DN3a stage cells. The transcript abundances of GATA3 (A), two Tcrb constant regions (B), and multiple V regions (C) were quantified as fragments per kilobase per million (FPKM). None of the Tcrb regions showed a statistically significant difference (FDR of <0.05) between Tg and control wild-type cells.

Article Snippet: Totals of 10 μg and 5 μg of nuclear extracts were electrophoresed on a 4-to-15% gradient precast SDS-polyacrylamide gel (Bio-Rad), transferred to a nitrocellulose membrane (Li-Cor), and first blotted by using a rabbit anti-GATA3 antibody (clone 11599 [ 32 ]).

Techniques: RNA Sequencing Assay

Journal: Molecular and Cellular Biology

Article Title: GATA3 Abundance Is a Critical Determinant of T Cell Receptor β Allelic Exclusion

doi: 10.1128/MCB.00052-17

Figure Lengend Snippet: Gene ontology (GO) analysis of genes that exhibit statistically significant differences between TgCd2-GATA3 and wild-type thymocytes. (A) From RNA-seq analysis of DN3a stage cells (conducted as described in the legend to Fig. 3), 171 genes were found to exhibit a statistically significant difference (FDR of <0.05) between the two genotypes. The 126 genes downregulated in Tg mice are shown in pink, and the 45 upregulated genes are shown in light blue. The Cpa3 gene is highlighted in dark blue. The Gata3 gene (highlighted in red), the abundance of which is not statistically significant (FDR = 0.249), is also shown. (B and C) Gene ontology analysis of these 126 and 45 genes was performed by using the DAVID functional annotation tool (41) (https://david.ncifcrf.gov/). The classification stringency was set to “highest.”

Article Snippet: Totals of 10 μg and 5 μg of nuclear extracts were electrophoresed on a 4-to-15% gradient precast SDS-polyacrylamide gel (Bio-Rad), transferred to a nitrocellulose membrane (Li-Cor), and first blotted by using a rabbit anti-GATA3 antibody (clone 11599 [ 32 ]).

Techniques: RNA Sequencing Assay, Functional Assay

Journal: Molecular and Cellular Biology

Article Title: GATA3 Abundance Is a Critical Determinant of T Cell Receptor β Allelic Exclusion

doi: 10.1128/MCB.00052-17

Figure Lengend Snippet: Genomic configuration of Tcrb alleles in individual DN4 stage thymocytes from wild-type or Gata3 mutant mice

Article Snippet: Totals of 10 μg and 5 μg of nuclear extracts were electrophoresed on a 4-to-15% gradient precast SDS-polyacrylamide gel (Bio-Rad), transferred to a nitrocellulose membrane (Li-Cor), and first blotted by using a rabbit anti-GATA3 antibody (clone 11599 [ 32 ]).

Techniques: Mutagenesis

Journal: Molecular and Cellular Biology

Article Title: GATA3 Abundance Is a Critical Determinant of T Cell Receptor β Allelic Exclusion

doi: 10.1128/MCB.00052-17

Figure Lengend Snippet: Tcrb VDJ rearrangement in mice that express superabundant GATA3. The numbers (green) next to each of the arrows represent the hypothetical cell numbers that would be predicted, under various conditions (bottom), at each developmental stage to obtain 100 surviving cells (shown in orange) after completion of thymic development. In marked contrast to wild-type mice, only 29% of DN4 stage thymocytes in TgCd2-GATA3 mice were of the Tcrb VDJ+/DJ genotype (Table 1). Instead, the vast majority of TgCd2-GATA3 DN4 stage cells (71%) had fully recombined (VDJ/VDJ) both Tcrb alleles. Importantly, we found that an extraordinary 19% of TgCd2-GATA3 DN4 stage cells bore DNA sequences that predicted two productively rearranged (VDJ+/VDJ+) Tcrb alleles, which is extremely rarely observed in wild-type mice (43). (A) One possible explanation (model 1) for the unexpected frequencies of DN4 Tcrb rearrangements in enforced GATA3 transgenic mice is that allelic exclusion is normal at the Tcrb loci at the DN3 stage (when the GATA3 protein abundance is normal, at 91% and 93% of the wild-type levels at the DN3a and DN3b stages, respectively), but later, at the DN4 stage, when the level of the GATA3 protein has increased (245% of the wild-type level) (Fig. 2C), the maintenance of allelic exclusion is forfeit in 50 to 70% of T cells, and the Tcrb genomic DNA now undergoes V-to-DJ rearrangement at the DN3b or DN4 stage, which has not been previously reported to occur in wild-type mice. (B) Alternative model (model 2). The human Cd2 regulatory sequences are active in DN1 (∼50%)-, DN2 (∼50%)-, DN3 (∼99%)-, DN4 (∼98%)-, and later-stage T cells based on an analysis of hCD2-GFP transgenic mice (38). In accord with those observations, we observed excess expression of GATA3 mRNA in TgCd2-GATA3 mice at the DN3a (151%) and DN3b (180%) stages compared to wild-type thymocytes (Fig. 2B). The abundance of the GATA3 protein in TgCd2-GATA3 mice increased by the DN4 stage, but a similar increase was not detectable at the DN3a or DN3b stage, when measured by the MFI of intracellular GATA3 staining (Fig. 2C). Therefore, we cannot exclude the possibility that a GATA3 mRNA-dependent mechanism compromises the initiation of allelic exclusion in 50 to 70% of DN3a cells (right) to generate VDJ+/VDJ+ cells in TgCd2-GATA3 mice. In either model, the excess expression of GATA3 predicts compromised allelic exclusion at the DN3a or DN4 stage. This altered regulatory potential of GATA3 supports the hypothesis that increased GATA3 expression partially caused by the Gata3 monoallelic-to-biallelic switch releases the second Tcrb locus from allelic exclusion (Fig. 1).

Article Snippet: Totals of 10 μg and 5 μg of nuclear extracts were electrophoresed on a 4-to-15% gradient precast SDS-polyacrylamide gel (Bio-Rad), transferred to a nitrocellulose membrane (Li-Cor), and first blotted by using a rabbit anti-GATA3 antibody (clone 11599 [ 32 ]).

Techniques: Transgenic Assay, Expressing, Staining

Journal: Molecular and Cellular Biology

Article Title: GATA3 Abundance Is a Critical Determinant of T Cell Receptor β Allelic Exclusion

doi: 10.1128/MCB.00052-17

Figure Lengend Snippet: Dual-TCRβ thymocytes are abundant in the thymi but not the spleens of TgCd2-GATA3 mice. (A) Representative flow cytometric analysis of Vβ14 (ordinate axis) versus a pool containing 11 different anti-Vβ antibodies (abscissa) (anti-Vβ2, -4, -5.1/5.2, -6, -7, -8, -9, -10b, -11, -12, and -13) from CD3+ thymocytes (top) or splenocytes (bottom) isolated from wild-type (Gata3+/+) (left) or TgCd2-GATA3 (right) animals. The percentages of cells expressing two different cell surface TCR Vβ+ complexes are shown in the insets. (B) Graphical summary of the frequencies of CD3+ thymocytes and splenocytes from wild-type (Gata3+/+) and TgCd2-GATA3 mice that express the indicated combinations of TCR Vβ (anti-Vβ14, -Vβ8, and -Vβ5.1/5.2 versus the Vβ pool containing anti-Vβ2, -4, -5.1/5.2, -6, -7, -8, -9, -10b, -11, -12, and -13; note that Vβ8 and Vβ5.1/5.2 were not included in the Vβ pool when Vβ8 and Vβ5.1/5.2 were used for individual staining, respectively) and Vα (anti-Vα2 versus the Vα pool, including anti-Vα3.2, -8.3, and -11.1/11.2) (see Table 3 for details on antibodies used). Each circle represents results for an individual animal from two independent experiments for a total of eight mice. The horizontal bar for each group indicates the average frequency. *, P < 0.05; NS, not significant.

Article Snippet: Totals of 10 μg and 5 μg of nuclear extracts were electrophoresed on a 4-to-15% gradient precast SDS-polyacrylamide gel (Bio-Rad), transferred to a nitrocellulose membrane (Li-Cor), and first blotted by using a rabbit anti-GATA3 antibody (clone 11599 [ 32 ]).

Techniques: Isolation, Expressing, Staining

Journal: Molecular and Cellular Biology

Article Title: GATA3 Abundance Is a Critical Determinant of T Cell Receptor β Allelic Exclusion

doi: 10.1128/MCB.00052-17

Figure Lengend Snippet: Antibodies used for flow cytometry c

Article Snippet: Totals of 10 μg and 5 μg of nuclear extracts were electrophoresed on a 4-to-15% gradient precast SDS-polyacrylamide gel (Bio-Rad), transferred to a nitrocellulose membrane (Li-Cor), and first blotted by using a rabbit anti-GATA3 antibody (clone 11599 [ 32 ]).

Techniques: Flow Cytometry, Conjugation Assay, Staining

Journal: Molecular and Cellular Biology

Article Title: GATA3 Abundance Is a Critical Determinant of T Cell Receptor β Allelic Exclusion

doi: 10.1128/MCB.00052-17

Figure Lengend Snippet: Wild-type and mutant DN4 stage thymocytes can be separated into two distinct pools based on the relative abundance of GATA3 expression. (A) Gata3g/+ DN4 stage thymocytes can be resolved into two distinct populations, one that does not express GFP and one that does (49), compared to DN4 stage thymocytes from Gata3+/+ mice. The middle and bottom panels depict the expression of the eGFP− and eGFP+ populations after resorting. (B) GATA3HI and GATA3LO DN4 stage populations in wild-type mice. Thymocytes isolated from wild-type mice were stained to identify DN4 stage-specific cell surface markers and then fixed for intracellular GATA3 staining or the IgG background. Total DN4-gated cells are shown at the top, and separate GATA3HI and GATA3LO flow-sorted cells (middle and bottom) were then reanalyzed by flow cytometry. A representative histogram is shown; this pattern is gender independent and was reproducible in at least 10 wild-type animals in five independent experiments (see Fig. 8C).

Article Snippet: Totals of 10 μg and 5 μg of nuclear extracts were electrophoresed on a 4-to-15% gradient precast SDS-polyacrylamide gel (Bio-Rad), transferred to a nitrocellulose membrane (Li-Cor), and first blotted by using a rabbit anti-GATA3 antibody (clone 11599 [ 32 ]).

Techniques: Mutagenesis, Expressing, Isolation, Staining, Flow Cytometry

Journal: Molecular and Cellular Biology

Article Title: GATA3 Abundance Is a Critical Determinant of T Cell Receptor β Allelic Exclusion

doi: 10.1128/MCB.00052-17

Figure Lengend Snippet: Profiles of GATA3 in wild-type and mutant cells. (A and B) Representative histograms of intracellular GATA3 protein abundance analyzed by flow cytometry in ETP, DN2, DN3a, DN3b, and DN4 stage thymocytes isolated from 5- to 8-week-old TgCd2-GATA3 mice (A) and wild-type (Gata3+/+) mice (B). Thymocytes were first stained for the cell surface markers used to distinguish between the various early developmental T cell stages (Materials and Methods) and then fixed and stained for intracellular GATA3 or IgG. (C) Gender-independent GATA3 protein expression in DN4 stage cells. Shown are intracellular GATA3 protein abundances in DN4 stage thymocytes from female (top) or male (bottom) wild-type animals. Eight individual animals were analyzed in seven independent experiments. (D) Quantification of GATA3 protein abundance in wild-type DN4 stage cells by flow cytometry as described above for panel C. The relative abundance of GATA3 was calculated based on the MFI of the GATA3HI population normalized to the GATA3LO population to eliminate the fluorescence intensity alteration due to flow cytometry settings that differ in independent experiments. Each circle represents results for an individual animal. Solid bars indicate the averages for each genotype. *, P < 0.05.

Article Snippet: Totals of 10 μg and 5 μg of nuclear extracts were electrophoresed on a 4-to-15% gradient precast SDS-polyacrylamide gel (Bio-Rad), transferred to a nitrocellulose membrane (Li-Cor), and first blotted by using a rabbit anti-GATA3 antibody (clone 11599 [ 32 ]).

Techniques: Mutagenesis, Flow Cytometry, Isolation, Staining, Expressing, Fluorescence

Journal: Molecular and Cellular Biology

Article Title: GATA3 Abundance Is a Critical Determinant of T Cell Receptor β Allelic Exclusion

doi: 10.1128/MCB.00052-17

Figure Lengend Snippet: Summary of mono- and biallelic GATA3 mRNA expression in single, staged adult thymocytes (GATA3 RT-PCR SNV sequence) in TCRα TCRβ or TCRβ transgenic mice a

Article Snippet: Totals of 10 μg and 5 μg of nuclear extracts were electrophoresed on a 4-to-15% gradient precast SDS-polyacrylamide gel (Bio-Rad), transferred to a nitrocellulose membrane (Li-Cor), and first blotted by using a rabbit anti-GATA3 antibody (clone 11599 [ 32 ]).

Techniques: Expressing, Sequencing, Transgenic Assay

Journal: Molecular and Cellular Biology

Article Title: GATA3 Abundance Is a Critical Determinant of T Cell Receptor β Allelic Exclusion

doi: 10.1128/MCB.00052-17

Figure Lengend Snippet: Relative expression levels of genes known to affect Tcrb allelic exclusion. Shown are data from qRT-PCR analysis of genes related to VDJ rearrangement and allelic exclusion at the Tcrb locus in DN3a, DN3b, and DN4 stage thymocytes isolated from TgCd2-GATA3 and control wild-type mice at 5 to 8 weeks of age. The mRNA expression levels were normalized to the values for beta-actin at each stage. The fold change relative to the wild type (set at 1) at each stage is shown. GATA3 expression represents total GATA3 transcripts regulated by hCd2 regulatory sequences plus endogenous GATA3 (same as shown in Fig. 2B). The data summarize the results of analyses of four mice of each genotype from two independent experiments. *, P < 0.05.

Article Snippet: Totals of 10 μg and 5 μg of nuclear extracts were electrophoresed on a 4-to-15% gradient precast SDS-polyacrylamide gel (Bio-Rad), transferred to a nitrocellulose membrane (Li-Cor), and first blotted by using a rabbit anti-GATA3 antibody (clone 11599 [ 32 ]).

Techniques: Expressing, Quantitative RT-PCR, Isolation

Journal: Molecular and Cellular Biology

Article Title: GATA3 Abundance Is a Critical Determinant of T Cell Receptor β Allelic Exclusion

doi: 10.1128/MCB.00052-17

Figure Lengend Snippet: Model for how altered GATA3 abundance regulates Tcrb VDJ rearrangement. The illustration summarizes how the GATA3 abundance might modulate Tcrb VDJ rearrangement: while approximately 90% of DN4 stage T cells that express less GATA3 (monoallelic Gata3 cells) bear a Tcrb VDJ+/DJ genotype, reciprocally, approximately 90% of DN4 stage cells that express higher levels of GATA3 (biallelic Gata3 cells) have rearranged both Tcrb alleles (Table 1). This led to the hypothesis that the GATA3 abundance must be quite precisely regulated in order to maintain allelic exclusion at the Tcrb locus and further suggested that the increase in the GATA3 abundance resulting at least partially from the Gata3 monoallelic-to-biallelic transcriptional switch might be responsible for the release of the second (only DJ rearranged) Tcrb locus from allelic exclusion.

Article Snippet: Totals of 10 μg and 5 μg of nuclear extracts were electrophoresed on a 4-to-15% gradient precast SDS-polyacrylamide gel (Bio-Rad), transferred to a nitrocellulose membrane (Li-Cor), and first blotted by using a rabbit anti-GATA3 antibody (clone 11599 [ 32 ]).

Techniques:

Journal: Molecular and Cellular Biology

Article Title: GATA3 Abundance Is a Critical Determinant of T Cell Receptor β Allelic Exclusion

doi: 10.1128/MCB.00052-17

Figure Lengend Snippet: GATA3 binding in the Tcrb locus. Short-read data (Sequence Read Archive [SRA] files) of GATA3 ChIP-seq experiments performed on DN and DP stage thymocytes (GEO accession number GSE20898) (58) were obtained from the public GEO database. The raw reads were aligned to mouse genome build mm10 by using DNASTAR software to generate wig files. The wig files were loaded into Integrated Genome Browser software version 9.0.0 (http://bioviz.org/igb/). The genome region around the Tcrb locus (chromosome 6, positions 39700001 to 42700000) is shown. y axis scales are set by value (minimum of 5 and maximum of 22) for both DN and DP data. The positions of the V, D, J, and C regions of the Tcrb gene as well as its enhancer (83) are depicted at the bottom.

Article Snippet: Totals of 10 μg and 5 μg of nuclear extracts were electrophoresed on a 4-to-15% gradient precast SDS-polyacrylamide gel (Bio-Rad), transferred to a nitrocellulose membrane (Li-Cor), and first blotted by using a rabbit anti-GATA3 antibody (clone 11599 [ 32 ]).

Techniques: Binding Assay, Sequencing, ChIP-sequencing, Software