Recombinant Mouse WT1 Protein

• Cat.No.: WT1-18590M
• Product Overview: Recombinant Mouse WT1 full length or partial length protein was expressed.

Specification

• Species: Mouse
• Source: Mammalian Cells
• Tag: His
• Form: Liquid or lyophilized powder
• Endotoxin: < 1.0 EU per μg of the protein as determined by the LAL method.
• Purity: >80%
• Notes: This item requires custom production and lead time is between 5-9 weeks. We can custom produce according to your specifications.
• Storage: Store it at +4 ºC for short term. For long term storage, store it at -20 ºC～-80 ºC.
• Storage Buffer: PBS buffer

Gene Information

• Gene Name: Wt1 Wilms tumor 1 homolog [ Mus musculus ]
• Official Symbol: WT1
• Gene ID: 22431
• mRNA Refseq: NM_144783.2
• Protein Refseq: NP_659032.3
• UniProt ID: P22561

Citation

WT1 Interacts with MAD2 and Regulates Mitotic Checkpoint Function

Journal: Nature communications    PubMed ID: 25232865    Data: 2014/8/5

Authors: Jayasha Shandilya, Eneda Toska, Stefan GE Roberts

Article Snippet:GST, GST-WT1 (and deletion mutants) were expressed in BL21-DE3 competent cells, and His-tagged human MAD2 (and mutant derivatives) were expressed in BL21-DE3(pLysS) competent cells followed by affinity purification as described before .GST, GST-WT1 (and deletion mutants) were expressed in BL21-DE3 competent cells, and His-tagged human MAD2 (and mutant derivatives) were expressed in BL21-DE3(pLysS) competent cells followed by affinity purification as described before .. Flag-tagged full length WT1 protein (residue 1–449, including exon 5/17AA and KTS) was purchased from Creative BioMart.. The MAD2-binding peptide 1 (MBP1) was purchased from Peptide 2.0 (sequence: SWYSYPPPQRAV); Control peptide sequence: CKATKDPSRVGDS.The MAD2-binding peptide 1 (MBP1) was purchased from Peptide 2.0 (sequence: SWYSYPPPQRAV); Control peptide sequence: CKATKDPSRVGDS.

(a) In vitro interaction assay was performed with either GST-WT1 (residues 245–297) or GST in the presence of full length His-MAD2. The interaction was analyzed by immunoblotting with anti-MAD2 antibody. Bound proteins were also resolved by SDS-PAGE and stained with Coomassie Brilliant Blue. (b) In vitro pulldown assay was also performed with either Flag-M2 magnetic beads alone or incubated with full length Flag-tagged WT1 protein in presence of His-MAD2. The interaction was analyzed by immunoblotting with anti-WT1 and anti-MAD2 antibodies (c) GST-interaction assay was carried out with WT1 containing the 17 amino acid insertion (residues 180-297, +17AA) or lacking it (residues 180-297, Δ17 AA) with His-MAD2. The interaction was analyzed by immunoblotting with anti-MAD2 antibody. Bound proteins were also resolved by SDS-PAGE and stained with Coomassie Brilliant Blue. (d) HeLa cells were transfected with GFP Vector, full length GFP-WT1 (?/?) or GFP-WT1 (?/+) isoforms and 48 hours later whole cell extracts were prepared followed by immunoprecipitation with anti-MAD2 antibodies. The immunoprecipitates were probed with anti-WT1 antibody. Blotting with anti-MAD2 antibody was performed as a control. (e) The MAD2 interaction with ectopically expressed GFP tagged full length WT1 (+/+) and WT1 (+/?) isoforms was analyzed as in part d (f) HeLa cells were transfected with pcDNA vector, pcDNA driving expression of WT1?/?DDS (R394X) or wild type WT1?/? followed by immunoprecipitation with anti-MAD2 antibodies. The immunoprecipitates were probed with anti-WT1 antibodies. Blotting with anti-MAD2 antibodies was performed as a control.

(a) GST-interaction assays were performed with the internal deletion mutants of GST-WT1 (schematic) with full length His-MAD2 and immunoblotted with anti-MAD2 and anti-GST antibodies. (b) Co-immunoprecipitation assays were carried out with whole cell extracts derived from HeLa cells that had been transfected with either GFP vector, GFP WT1 (?/?) or GFP-WT1 (?/?Δ4) using anti-MAD2 or anti-WT1 antibodies. (c) Co-immunofluorescence analysis was carried out in WiT49 cells with anti-MAD2 and anti-WT1 antibodies at the pro-metaphase stage of mitosis. DNA was stained with Hoechst. The scale bar is 10 microns. (d) M15, WiT49 and K562 cells were treated with 60 ng/ml nocodazole for 24 hours, whole cell extracts prepared and immunoprecipitation performed with anti-MAD2 antibodies followed by immunoblotting with WT1 antibody. Input is 10% of the total whole cell extract.

(a) GST-interaction assays were carried out with WT1 (residues 245–297) and full length (FL) His-MAD2 alone and also with MAD2 pre-incubated with either 40μM of MAD2-binding peptide 1 (MBP1), or a control peptide. The pulldown complexes were resolved by SDS-PAGE and immunoblotted with anti-MAD2 antibodies. (b) In vitro pulldown assays were performed with either Flag-M2 magnetic beads alone or full length Flag-tagged WT1 protein in the presence of Full length His-MAD2 (pre-incubated with MBP1) or the MAD2 delC mutant. The interaction was analyzed by immunoblotting with anti-WT1 and anti-MAD2 antibodies. (c) GST-interaction assays were carried out with His-MAD2, MAD2 L13A and MAD2-C-terminal deletion mutant (Del-C). Immunoblotting was performed using anti-MAD2 and anti- GST antibodies. (d) GST-interaction assays were carried out with His-MAD2 + MBP1, MAD2-Del-C, and MAD2 dimerization mutant, R133A. Immunoblotting was performed using anti-MAD2 and anti-GST antibodies. (e) Pull down assays were performed with full length Flag-WT1 protein and the different MAD2 point mutant derivatives. Immunoblotting was performed using anti-MAD2 and anti-WT1 antibodies. Input is 20% of the different MAD2 proteins used for interaction analysis.