rabbit polyclonal anti sox6  (Atlas Antibodies)

Journal: Cell reports

Article Title: Systematic multi-omics cell line profiling uncovers principles of Ewing sarcoma fusion oncogene-mediated gene regulation

doi: 10.1016/j.celrep.2022.111761

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-SOX6 , Atlas Antibodies, Stockholm, Sweden , Cat#HPA003908; RRID: AB_1080063.

Techniques: Plasmid Preparation, Polymer, Virus, In Vivo, Recombinant, Reverse Transcription, DNA Methylation Assay, Knockdown, Gene Expression, Microarray, shRNA, Software

Journal: Skeletal Muscle

Article Title: Trip12, a HECT domain E3 ubiquitin ligase, targets Sox6 for proteasomal degradation and affects fiber type-specific gene expression in muscle cells

doi: 10.1186/2044-5040-3-11

Figure Lengend Snippet: SOX6 coiled-coil domain physically interacts with TRIP12 HECT domain. ( A ) Schematic representation of the human SOX6 protein . The coiled-coil (CC) domain containing the leucine-zipper (LZ) motif and Q-box (aa 143–304) was used as bait for screening a human heart cDNA library. The amino acid sequence of the CC domain is 100% conserved between mice and humans. ( B ) Schematic representation of the full-length human TRIP12 protein. The partial TRIP12 cDNA clone identified by yeast hybrid screening contained the most C-terminal HECT domain (aa 1614–2040). ( C ) Diagrams of the bait and prey expression vectors used for Co-IP. The numbers indicate the amino acid regions used for each vector construct. The SOX6 CC-domain was tagged with c-Myc at the C-terminus. The TRIP12 HECT domain was tagged with HSV at the C-terminus. ( D ) Co-IP assays were performed using HEK293 cell lysate transfected with the bait and prey expression vectors depicted in ( C ). Input lanes contained 2% (10 μg) of un-manipulated lysate. Rabbit polyclonal antibodies used for pull down are listed under IP. Mouse monoclonal antibodies used for Western blot (WB) are indicated below each panel. GST antibody was used as a negative control.

Article Snippet: IP and Western blotting was performed with Rabbit TrueBlot Set (Rockland Immunochemicals, Inc., Gilbertsville, PA, USA) using 300 μg of nuclear protein and 2 μg of rabbit polyclonal antibodies against Sox6 (ab30455, Abcam), TRIP12 (A301-814A, Bethyl Laboratories, Inc.), and GST (ab9085, Abcam) according to the manufacturer’s instructions.

Techniques: cDNA Library Assay, Sequencing, Expressing, Co-Immunoprecipitation Assay, Plasmid Preparation, Construct, Transfection, Western Blot, Negative Control

Journal: Skeletal Muscle

Article Title: Trip12, a HECT domain E3 ubiquitin ligase, targets Sox6 for proteasomal degradation and affects fiber type-specific gene expression in muscle cells

doi: 10.1186/2044-5040-3-11

Figure Lengend Snippet: Trip12 protein expression in adult mouse tissues and interaction between endogenous Sox6 and Trip12 proteins. ( A ) Western blot analysis was performed to determine Trip12 protein expression in adult mouse tissues. Each lane contained 30 μg of protein except for the C12C12 samples, which contained 15 μg of protein per lane. Relevant protein size markers (kDa) are indicated to the left. β-actin was used as a loading control. ( B ) Co-IP of endogenous Sox6 and Trip12 proteins using nuclear fractions of differentiating C2C12 cells. Input lane contained 5% (15 μg) of pre-cleared nuclear protein. Antibodies used for pull down are listed under IP, and antibodies used for Western blot (WB) are indicated below each panel. GST antibody was used as a negative control.

Article Snippet: IP and Western blotting was performed with Rabbit TrueBlot Set (Rockland Immunochemicals, Inc., Gilbertsville, PA, USA) using 300 μg of nuclear protein and 2 μg of rabbit polyclonal antibodies against Sox6 (ab30455, Abcam), TRIP12 (A301-814A, Bethyl Laboratories, Inc.), and GST (ab9085, Abcam) according to the manufacturer’s instructions.

Techniques: Expressing, Western Blot, Co-Immunoprecipitation Assay, Negative Control

Journal: Skeletal Muscle

Article Title: Trip12, a HECT domain E3 ubiquitin ligase, targets Sox6 for proteasomal degradation and affects fiber type-specific gene expression in muscle cells

doi: 10.1186/2044-5040-3-11

Figure Lengend Snippet: TRIP12 ubiquitinates Sox6 in vitro and in vivo . ( A ) In vitro ubiquitination of SOX6 protein was performed using purified SOX6-myc as the substrate and TRIP12 along with the combination of enzymes as indicated in the figure (see the Methods section for details). Lanes 4–6 contain 3 μg of TRIP12-HSV. Western blot (WB) was performed using c-Myc antibody to detect the degree of ubiquitination of Sox6-myc. Asterisk indicates non-specific bands detected in all reactions. ( B ) Sox6 is ubiquitinated by TRIP12 in vivo . HEK293 cells were cotransfected with plasmid DNAs encoding HA-Ub, Sox6-FLAG, and increasing amounts (0.5, 1, and 1.5 μg) of TRIP12-HSV, and lysates were immunoprecipitated (IP) with anti-DYKDDDDK (FLAG) antibody, and then processed for Western blotting (WB) using anti-HA antibody. Asterisk indicates non-specific bands detected in all IP samples, although it is possible that these bands also contain ubiquitinated Sox6-FLAG protein of lower molecular weights (with ~1 to 4 ubiquitin moieties) in Sox6-FLAG-transfected samples. The same membrane was subsequently incubated with anti-DYKDDDDK (FLAG) antibody to detect Sox6-FLAG protein; 10 μg (2%) of input protein samples (lysates) was also subjected to Western blotting using anti-TRIP 12 antibody to detect both endogenous TRIP12 and overexpressed TRIP12-HSV proteins.

Article Snippet: IP and Western blotting was performed with Rabbit TrueBlot Set (Rockland Immunochemicals, Inc., Gilbertsville, PA, USA) using 300 μg of nuclear protein and 2 μg of rabbit polyclonal antibodies against Sox6 (ab30455, Abcam), TRIP12 (A301-814A, Bethyl Laboratories, Inc.), and GST (ab9085, Abcam) according to the manufacturer’s instructions.

Techniques: In Vitro, In Vivo, Purification, Western Blot, Plasmid Preparation, Immunoprecipitation, Transfection, Incubation

Journal: Skeletal Muscle

Article Title: Trip12, a HECT domain E3 ubiquitin ligase, targets Sox6 for proteasomal degradation and affects fiber type-specific gene expression in muscle cells

doi: 10.1186/2044-5040-3-11

Figure Lengend Snippet: Trip12 controls Sox6 protein level in C2C12 cells. (A) siRNA-mediated knockdown of Trip12 resulted in an increase of Sox6 protein levels in C2C12 cells. C2C12 cells were transfected with siRNA for either EGFP (negative control) or Trip12 in triplicate, and lysates were analyzed by Western blotting using a 7.5% gel. (B) Densitometric analysis of the Western blot in ( A ) shows approximately 3-fold increase in the Sox6 protein level in Trip12 siRNA-treated cells, while the Tbp protein level was not affected. Data are normalized for those from EGFP siRNA-transfected cells and represented as mean ± SD ( n = 3). (C) Trip12 knockdown lowered mRNA level of Myh7 , a known Sox6 target. Total RNA was extracted from mock- or siRNA-transfected C2C12 cells, and mRNA levels of Trip12 and Myh7 were quantified by reverse transcription-quantitative PCR (RT-qPCR). Data are normalized for those from EGFP siRNA-transfected cells and represented as mean ± SD ( n = 3). ** p < 0.005.

Article Snippet: IP and Western blotting was performed with Rabbit TrueBlot Set (Rockland Immunochemicals, Inc., Gilbertsville, PA, USA) using 300 μg of nuclear protein and 2 μg of rabbit polyclonal antibodies against Sox6 (ab30455, Abcam), TRIP12 (A301-814A, Bethyl Laboratories, Inc.), and GST (ab9085, Abcam) according to the manufacturer’s instructions.

Techniques: Transfection, Negative Control, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Skeletal Muscle

Article Title: Trip12, a HECT domain E3 ubiquitin ligase, targets Sox6 for proteasomal degradation and affects fiber type-specific gene expression in muscle cells

doi: 10.1186/2044-5040-3-11

Figure Lengend Snippet: Sox6 is degraded by proteasome in C2C12 cells. ( A ) Time course of Trip12, Sox6, and Tbp protein levels in C2C12 cells in the presence of cycloheximde (CHX) analyzed by Western blotting using a 4-15% gradient gel ( n = 3). ( B ) Densitometric analysis of the Western blot in ( A ). Band intensity of each protein was normalized to that of 0 h in CHX and represented as mean ± SD ( n = 3). ( C ) Validation of Psmd1 knockdown by siRNA. Total RNA was extracted from mock- or siRNA-transfected C2C12 cells, and the mRNA level of Psmd1 (the gene encoding a regulatory subunit of the 26S proteasome) was quantified by RT-qPCR. Data are normalized to EGFP siRNA-transfected cells and represented as mean ± SD ( n = 3). ( D ) A Western blot using a 7.5% gel showing an increase in the Sox6 protein level in Psmd1 siRNA-transfected C2C12 cells. ( E ) Densitometric analysis of Western blotting results shows a ~4 fold increase in the Sox6 protein level in Psmd1 siRNA-treated C2C12 cells. A smaller increase was observed for Tbp protein. Data are normalized for those from EGFP siRNA-transfected cells and represented as mean ± SD ( n = 3). ( F ) Psmd1 knockdown reduced the mRNA level of Myh7 , a known target of Sox6. Data are normalized to EGFP siRNA-transfected cells and represented as mean ± SD ( n = 3). ** p < 0.005.

Article Snippet: IP and Western blotting was performed with Rabbit TrueBlot Set (Rockland Immunochemicals, Inc., Gilbertsville, PA, USA) using 300 μg of nuclear protein and 2 μg of rabbit polyclonal antibodies against Sox6 (ab30455, Abcam), TRIP12 (A301-814A, Bethyl Laboratories, Inc.), and GST (ab9085, Abcam) according to the manufacturer’s instructions.

Techniques: Western Blot, Transfection, Quantitative RT-PCR

Journal: Nature communications

Article Title: Oncogenic hijacking of a developmental transcription factor evokes vulnerability toward oxidative stress in Ewing sarcoma.

doi: 10.1038/s41467-020-16244-2

Figure Lengend Snippet: Fig. 1 SOX6 is highly but variably expressed in EwS. a SOX6 expression levels (Affymetrix microarrays) in EwS tumors, nine additional sarcoma or pediatric tumor entities, and 18 normal tissue types. Data are represented as dot plots, horizontal bars represent medians. The number of biologically independent samples per group (n) is given in parentheses. ASPS, alveolar soft part sarcoma; GIST, gastrointestinal stromal tumor. b Validation of SOX6 protein expression by IHC in the same tissue types as shown in (a). Immuno Reactive scores (IRS) are presented as dot plots. Horizontal bars represent medians. The number of biologically independent samples per group (n) is given in parentheses. c Representative micrographs of the IHC stains from samples of the same tumor entities or normal tissue types indicated in (b); scale bars = 20 µm. Source data are provided as a Source Data file.

Article Snippet: The slides were stained with either polyclonal anti-SOX6 antibody raised in rabbit (1:1600; HPA003908, Atlas Antibodies, Sweden) or with monoclonal anti-Ki67 raised in rabbit (1:200, 275R-15, Cell Marque/Sigma-Aldrich) or with a monoclonal anti-TXNIP antibody raised in rabbit (1:250; EPR14774, Abcam) for 60 min at RT, followed by a monoclonal secondary horseradish peroxidase (HRP)-coupled horse-anti-rabbit antibody (ImmPRESS Reagent Kit, MP7401, Vector Laboratories, Germany).

Techniques: Expressing, Biomarker Discovery

Journal: Nature communications

Article Title: Oncogenic hijacking of a developmental transcription factor evokes vulnerability toward oxidative stress in Ewing sarcoma.

doi: 10.1038/s41467-020-16244-2

Figure Lengend Snippet: Fig. 2 EWSR1-FLI1 induces SOX6 expression via an intronic GGAA-mSat. a EWSR1-FLI1 and SOX6 expression (qRT-PCR) in A673/TR/shEF1 cells after addition of Dox. Horizontal bars represent means, n = 3 biologically independent experiments for time points 0 h, 72 h and 168 h, n = 6 biologically independent experiments for the time point 96 h. P values determined by two-sided Mann–Whitney test. EWSR1-FLI1: 72 h (P = 0.012), 96 h (P = 0.0007), 168 h (P = 0.012); SOX6: 72 h (P = 0.017), 96 h (P = 0.001), 168 h (P = 0.017). b Left: EWSR1-FLI1 and SOX6 expression (Affymetrix microarrays) in A673/ TR/shEF1 xenografts after 96 h of Dox-treatment. Horizontal bars represent means, n = 3 biologically independent xenografts per group. P value determined via two-sided independent one-sample t-test (EWSR1-FLI1: P = 0.002, SOX6 P = 0.028). Right: Representative immunohistological staining for (EWSR1-)FLI1 and SOX6. Scale bar = 20 µm. c SOX6 expression (Affymetrix microarrays) in embryoid bodies after ectopic EWSR1-FLI1 expression. Horizontal bars represent means, n = 3 biologically independent samples per group. P value determined via unpaired two-sided t-test with Welch’s correction (P = 0.002). d Integrated genomic view of DNAse I hypersensitivity (HS) and ChIP-Seq data for EWSR1-FLI1 and H3K27ac in EwS cells transfected with shRNA against EWSR1-FLI1 (shEF1) or control shRNA (shGFP). e Relative enhancer activity of the SOX6-associated GGAA-mSat in A673/ TR/shEF1 cells (−/+ Dox). Horizontal bars represent means, n = 4 biologically independent experiments. P value determined via two-sided Mann–Whitney test (P = 0.029). f Correlation of the average enhancer activity of both alleles of the SOX6-associated GGAA-mSat and the average SOX6 levels across eight EwS cell lines (TC-32 set as reference). The color code indicates the average number of consecutive GGAA-repeats of both alleles. P value determined via two-tailed Pearson correlation test, n = 4 biologically independent experiments. All error bars represent SEM. ***P < 0.001, **P < 0.01, *P < 0.05. Source data are provided as a Source Data file.

Article Snippet: The slides were stained with either polyclonal anti-SOX6 antibody raised in rabbit (1:1600; HPA003908, Atlas Antibodies, Sweden) or with monoclonal anti-Ki67 raised in rabbit (1:200, 275R-15, Cell Marque/Sigma-Aldrich) or with a monoclonal anti-TXNIP antibody raised in rabbit (1:250; EPR14774, Abcam) for 60 min at RT, followed by a monoclonal secondary horseradish peroxidase (HRP)-coupled horse-anti-rabbit antibody (ImmPRESS Reagent Kit, MP7401, Vector Laboratories, Germany).

Techniques: Expressing, Quantitative RT-PCR, MANN-WHITNEY, Staining, ChIP-sequencing, Transfection, shRNA, Control, Activity Assay, Two Tailed Test