Recombinant Human SOX10 protein, MYC/DDK-tagged

• Cat.No.: SOX10-1565H
• Product Overview: Recombinant Human SOX10, fussed with MYC/DDK tag at C-terminal, was expressed in HEK293 cells.

Specification

• Species: Human
• Source: HEK293
• Tag: DDK&Myc
• Description: This gene encodes an integral membrane protein that is required for cytokine-induced regulation of the tight junction paracellular permeability barrier. Mutations in this gene are thought to be a cause of band-like calcification with simplified gyration and polymicrogyria (BLC-PMG), an autosomal recessive neurologic disorder that is also known as pseudo-TORCH syndrome. Alternative splicing results in multiple transcript variants. A related pseudogene is present 1.5 Mb downstream on the q arm of chromosome 5.
• Form: 25 mM Tris.HCl, pH 7.3, 100 mM glycine, 10% glycerol.
• Molecular Mass: 49.7 kDa
• Purity: > 80% as determined by SDS-PAGE and Coomassie blue staining
• Notes: Recombinant protein was captured through anti-DDK affinity column followed by conventional chromatography steps.
• Concentration: >50 ug/mL as determined by microplate BCA method
• Publications:
  Temporal activation of WNT/β-catenin signaling is sufficient to inhibit SOX10 expression and block melanoma growth (2020)

Gene Information

• Gene Name: SOX10 SRY (sex determining region Y)-box 10 [ Homo sapiens ]
• Official Symbol: SOX10
• Synonyms: SOX10; SRY (sex determining region Y)-box 10; transcription factor SOX-10; DOM; dominant megacolon; mouse; human homolog of; WS2E; WS4; SRY-related HMG-box gene 10; dominant megacolon, mouse, human homolog of; PCWH; WS4C; MGC15649
• Gene ID: 6663
• mRNA Refseq: NM_006941
• Protein Refseq: NP_008872
• MIM: 602229
• UniProt ID: P56693
• Chromosome Location: 22q13.1
• Function: DNA binding; RNA polymerase II core promoter proximal region sequence-specific DNA binding; RNA polymerase II distal enhancer sequence-specific DNA binding; chromatin binding; identical protein binding; protein binding; transcription coactivator activity; transcription regulatory region DNA binding

Citation

Temporal activation of WNT/β-catenin signaling is sufficient to inhibit SOX10 expression and block melanoma growth

Journal: Oncogene    PubMed ID: 32238882    Data: 2020/4/1

Authors: Rexhep Uka, Christian Britschgi, Olga Shakhova

Article Snippet:To identify novel SOX10-interacting proteins, a M010817 WCE was analyzed by nLc Orbitrap MS at CovalX (CovalX AG, Zürich, Switzerland).To identify novel SOX10-interacting proteins, a M010817 WCE was analyzed by nLc Orbitrap MS at CovalX (CovalX AG, Zürich, Switzerland).. M010817 were lysed in NE buffer (50 mM Tris pH 7.5, 150 mM KCl, 0.2 mM EDTA, 5 mM MgCl 2 , 20% Glycerol, 0.5 mM DTT) and then sent to CovalX for further analysis, along with purified SOX10 protein in a buffer of 25 mM Tris pH 7.3, 100 mM glycine, 10% glycerol, obtained from Creative BioMart (Creative BioMart Inc, Shirley, NY, USA).. Procedure at CovalX were as follows:Procedure at CovalX were as follows:

a Representative images of immunohistochemical stainings of human melanoma patient derived biopsies ( n = 13) before (left panel) and after (right panel) combined BRAF and MEK inhibitor treatment stained for SOX10 (red), pERK (red) and Pan Melanoma (PanMel), a cocktail of Melanoma antigen recognized by T cells-1 (MART-1) and Tyrosinase (green). Nuclei were counterstained with DAPI (blue). b Quantification of SOX10 expression levels pre- and post-BRAF and MEK inhibitor treatment. c Quantification of pERK expression levels pre- and post-BRAF and MEK inhibitor treatment. d Representative western blot for the indicated proteins in four different human melanoma cell lines. Levels of SOX10 expression are not decreased upon treatment with BRAF inhibitors (vemurafenib and dabrafenib, each 1 μM) or MEK inhibitor (selumetinib, 1 μM) after 24 h neither in single, nor in double treatments (vemurafenib and selumetinib). In MAPK inhibitor sensitive cell lines (M98, M00) pERK levels are not detectable anymore, which indicates the correct function of the drugs, whereas in resistant cell cultures (M11, M12) pERK levels were not affected ( n = 3). In b and c data represent mean ± s.d. Statistical significance was determined by paired, two-tailed Student’s t test. * P < 0.05, ** P < 0.01, *** P < 0.001 Western blots shown in d are representative. In each panel, n indicates the number of independent experiments performed.

a Schematic representation showing the nLc Orbitrap mass spectrometry (MS) analysis to identify SOX10-interacting proteins. M010817 whole cell extract (WCE) was analyzed performing nLc Orbitrap MS at CovalX. b Interaction of SOX10 and β-catenin was confirmed by co-immunoprecipitation using an anti-HA antibody for the pull down. c Representative immunocytochemical stainings indicating the subcellular localization of SOX10 (in red) and actin cytoskeleton (in green) in the MAPK inhibitor sensitive (M98, upper panel) and in the resistant human melanoma cell line M11 (lower panel) in presence of DMSO (left panel) or CHIR99021 (for 24 h, 6 μM) (right panel). Inserts show higher magnification of SOX10 immunostainings ( n = 3). d A representative western blot of the indicated proteins in presence or absence of the GSK3 α/β inhibitor CHIR99021 (for 24 h at 6 μM) in patient-derived melanoma cell cultures sensitive (M98, M00) against targeted MAPK inhibitors or resistant (M11, M12) ( n = 5). e A representative western blot for the indicated proteins in presence or absence of the GSK3 α/β inhibitor LY2090314 (for 24 h at 100 nM) in patient-derived melanoma cell cultures sensitive (M01 and M98) or resistant against MAPK inhibitors (M12 and M11) ( n = 3). f A representative western blot for the indicated proteins of M11 human melanoma cell culture stably expressing shCTNNB1 constructs cultured in presence or absence of CHIR99021 for 24 h, 6 μM ( n = 3). g On the left, representative western blot of the indicated proteins in the MAPK inhibitor resistant human melanoma cell culture M11 transiently expressing wild type β-catenin or a stabilizing mutated form of β-catenin (S33Y). Empty pCDNA3 was used as control (cnt). β-actin was used as loading control. On the right, SOX10 mRNA expression levels normalized to GAPDH in the MAPK inhibitor resistant human melanoma cell culture M11 transiently expressing wild type β-catenin or a stabilized mutated form of β-catenin (S33Y) ( n = 3). Data represent mean ± s.d. In d and e , β-actin was used as loading control. In f and g , GAPDH was used as loading control. In each panel, n indicates the number of independent experiments performed.

a Representative western blots of the indicated proteins in the MAPK inhibitor resistant human melanoma cell culture M12, at different timepoints of CHIR99021 (6 μM) treatment (timepoints are shown directly in the figure). GAPDH was used as loading control ( n = 3). b RNA levels in MAPK inhibitor sensitive (M98) human melanoma cell culture (ΔCt to GAPDH expression) after 4, 8, and 12 h of CHIR99021 (6 μM) treatment are shown compared with DMSO (ctrl) ( n = 3). c Representative western blot of the indicated proteins in the MAPK inhibitor sensitive human melanoma cell culture (M98). A representative western blot for the indicated proteins of M98 human melanoma cell culture stably expressing shSOX10 or shMITF constructs cultured in presence or absence of CHIR99021 for 24 h, 6 μM. Nssh represents the scrambled shRNA used as control construct ( n = 3). d Representative western blot of the indicated proteins in presence of CHIR99021 (6 μM) and/or MG-132 (20 μM) (in single or double treatments) for 16 h. GAPDH was used as a loading control ( n = 3). e Heatmap demonstrating the gene expression of SOX10 and Wnt target genes of M98 and M00 (MAPK inhibitor sensitive cell cultures) and M11 (resistant to MAPK inhibitor) in either presence or absence of CHIR99021. Data on the heat map represent log2 of sequencing reads assigned to genes. Statistical significance was determined by unpaired, two-tailed Student’s t test in b . * P < 0.05, ** P < 0.01, *** P < 0.001. In each panel, n indicates the number of independent experiments performed.