Recombinant Human X-linked Inhibitor Of Apoptosis, GST-tagged

• Cat.No.: XIAP-1503H
• Product Overview: Recombinant full-length human XIAP was expressed by baculovirus inSf9insect cells using a C-terminal GST tag. MW = 84 kDa.

Specification

• Species: Human
• Source: Sf9 Cells
• Tag: GST
• Description: XIAP is a member of a family of proteins which inhibit apoptosis and act as mammalian cell-death suppressors. XIAP inhibits at least 2 members of the caspase family of cell-death proteases, caspase-3 and caspase-7. In addition, XIAP inhibits apoptosis induced by menadione, a potent inducer of free radicals, and ICE. Furthermore, XIAP can mediate protection of cells from apoptosis by utilizing both a JNK1 activation pathway that involves ILPIP and a caspase inhibition pathway that is independent of ILPIP. Disruption of the XIAP gene in human colon cancer cells can enhance sensitivity of these cells to exogenously added TRAIL suggesting that XIAP is a nonredundant modulator of TRAIL-mediated apoptosis.
• Sequence: Full-length.
• Applications: Western Blot.
• Storage And Stability: Store product at -70℃. For optimal storage, aliquot target into smaller quantities after centrifugation and store at recommended temperature. For most favorable performance, avoid repeated handling and multiple freeze/thaw cycles.

Gene Information

• Gene Name: XIAP X-linked inhibitor of apoptosis [ Homo sapiens ]
• Synonyms: XIAP; X-linked inhibitor of apoptosis; API3; ILP1; MIHA; XLP2; BIRC4; baculoviral IAP repeat-containing protein 4; OTTHUMP00000196392; apoptosis inhibitor 3; baculoviral IAP repeat-containing 4; E3 ubiquitin-protein ligase XIAP; Inhibitor of apoptosis protein 3; X-linked inhibitor of apoptosis protein; X-linked IAP; IAP-like protein; HILP; IAP3; EC 6.3.2.-; OTTHUMP00000023975; hILP
• Gene ID: 331
• mRNA Refseq: NM_001167
• Protein Refseq: NP_001158
• MIM: 300079
• UniProt ID: P98170
• Chromosome Location: Xq25
• Pathway: Apoptosis; Focal adhesion; NOD-like receptor signaling pathway; Pathways in cancer; Small cell lung cancer; Ubiquitin mediated proteolysis
• Function: caspase inhibitor activity; ligase activity; metal ion binding; peptidase inhibitor activity; protein binding; zinc ion binding

Citation

Mutual regulation between OGT and XIAP to control colon cancer cell growth and invasion

Journal: Cell Death & Disease    PubMed ID: 32994395    Data: 2020/9/29

Authors: Hyeon Gyu Seo, Han Byeol Kim, Jin Won Cho

Article Snippet:Recombinant His-Ubiquitin protein and His-UCHL5 were purchased from R&D systems (Minneapolis, MN, USA) and LSBio (Seattle, WA, USA), respectively.Recombinant His-Ubiquitin protein and His-UCHL5 were purchased from R&D systems (Minneapolis, MN, USA) and LSBio (Seattle, WA, USA), respectively.. Recombinant GST-XIAP was purchased from Creative BioMart (Shirley, NY, USA).. Glutathione agarose and Ni-NTA agarose were purchased from Qiagen (Hilden, Germany).Glutathione agarose and Ni-NTA agarose were purchased from Qiagen (Hilden, Germany).

a HCT16 cell lysates were incubated with immobilized recombinant GST-OGT fusion protein (residues 1–1036). GST-OGT was precipitated and the associated XIAP was detected by western blotting with α-XIAP antibodies. GST and GST-OGT were detected by Coomassie brilliant blue staining to verify the amount of protein used in the assay. b HCT116 cells transiently overexpressing Flag-tagged XIAP were immunoprecipitated with α-Flag or IgG control antibodies. Co-immunoprecipitated endogenous OGT was detected by α-OGT antibodies. The same membrane was re-probed with α-XIAP antibodies. Total lysates were blotted with α-OGT and α-XIAP antibodies. c HCT116 cells were transfected with Flag-tagged OGT and immunoprecipitated with α-Flag or IgG control antibodies. Bound endogenous XIAP was detected by α-XIAP antibodies. The same membrane was re-probed with α-OGT antibodies. Total lysates were blotted with α-OGT and α-XIAP antibodies. d OGT in vivo ubiquitination assay. Expression vectors encoding Flag-OGT and HA-ubiquitin (Ub) were transfected into HCT116 cells transiently overexpressing Myc-XIAP as indicated. The cells were treated with 20 μM of MG132 for 4 hours (h) before they were harvested. After being immunoprecipitated with α-Flag antibodies, the ubiquitination of OGT was analyzed by immunoblotting with α-HA antibodies. The same membrane was re-probed with α-Flag antibodies. Equal amounts of total lysates were subjected to immunoblotting with the indicated antibodies. e OGT in vitro ubiquitination assay. GST-OGT was incubated with His-XIAP, HA-Ub, E1, E2, and ATP as indicated. After GST pull-down under denaturing conditions with a buffer containing 2% SDS, the ubiquitination of OGT was analyzed by immunoblotting with α-HA antibodies. The same membrane was re-probed with α-GST antibodies. Equal amounts of XIAP in the reaction were immunoblotted with α-His antibodies. f HCT116 WT or HCT116 XIAP KO cells were transfected with Flag-OGT and treated with 20 μM of MG132 for 4 h. The ubiquitination of immunoprecipitated OGT was analyzed by immunoblotting with α-Ub antibodies. The same membrane was re-probed with α-Flag antibodies. Equal amounts of total lysates were subjected to immunoblotting as indicated. β-actin was used as a loading control. All data are representative of at least three independent experiments.

a HCT116 cells were either treated with 1 μM of Thiamet-G for 4 h or not treated with any Thiamet-G. Cell lysates were then subjected to sWGA lectin affinity purification and analyzed with Western blotting for the presence of endogenous XIAP. As a control, 20 mM of the monosaccharide inhibitor GlcNAc was added during sWGA lectin affinity purification. Relative O-GlcNAcylated XIAP levels were plotted ( n = 5 per condition). b HCT116 cells were either treated with 1 μM of Thiamet-G for 4 h or not treated with any Thiamet-G. Immunoprecipitated endogenous XIAP was detected by α-O-GlcNAc antibodies. c Expression vectors encoding Flag-OGT were transiently transfected into HEK293 cells and immunoprecipitated Flag-XIAP was subjected to MS analysis. The CTD MS/MS spectrum of residues 406–419, O-GlcNAcylated XIAP peptides, with the doubly charged precursor ion m/z 851.461426 (M + 2H) 2+ is shown. The b- and y-type product ions were assigned. d Identification of the O-GlcNAc modification sites of XIAP by in vitro glycosylation assay. Purified WT or mutant His-tagged XIAP were used as substrates. O-GlcNAc-modified XIAP was analyzed by α-O-GlcNAc antibodies. The same membrane was re-probed with α-XIAP antibodies. Immunoblotting with α-GST antibodies was conducted to ensure that there was an equal amount of OGT. e Flag-XIAP WT, XIAP S406A, or XIAP ΔRING mutants were transiently overexpressed in HCT116 XIAP KO cells. XIAP WT and XIAP mutants were immunoprecipitated with α-Flag antibodies and blotted with α-O-GlcNAc and α-XIAP antibodies. Equal amounts of total lysates were subjected to immunoblotting with antibodies as indicated. β-actin or GAPDH was used as a loading control. Data are presented as means ± SD of at least three independent experiments. Statistical significance was determined using one-way analysis of variance. * P < 0.05, *** P < 0.001.

a Expression vectors encoding Flag-OGT and HA-Ub were transfected into HCT116 cells transiently overexpressing Myc-XIAP WT and Myc-XIAP S406A as indicated. Cells were treated with 10 μM of MG132 for 4 h before being harvested. After immunoprecipitation with α-Flag antibodies, the ubiquitination of OGT was analyzed by immunoblotting with α-HA antibodies. The same membrane was re-probed with α-Flag antibody. Equal amounts of total lysates were subjected to immunoblotting with the indicated antibodies. b GST-OGT was incubated with HA-Ub, E1, E2, ATP, His-UCHL5, and either His-XIAP WT or His-XIAP S406A in the presence of UDP-GlcNAc as indicated. After GST pull-down was conducted under denaturing conditions with a buffer containing 2% SDS, the ubiquitination of OGT was detected by immunoblotting with α-HA antibodies. The same membrane was re-probed with α-GST antibodies. The amounts of XIAP and UCHL5 in the reaction were detected with α-XIAP antibody and α-His antibodies, respectively. c Expression vectors encoding Flag-TAK1 and HA-Ub were transiently transfected into HCT116 cells overexpressing Myc-XIAP as indicated. Cells were treated with 10 μM of MG132 for 4 h before being harvested. After immunoprecipitation with α-Flag antibodies, the ubiquitination of TAK1 was analyzed by immunoblotting with α-HA antibodies. The same membrane was re-probed with α-Flag antibodies. Equal amounts of total lysates were subjected to immunoblotting with the indicated antibodies. d Expression vectors encoding Myc-Cdc42 and HA-Ub were transiently transfected into HCT116 cells overexpressing Flag-XIAP as indicated. Before harvest, cells were treated with 20 μM of MG132 for 4 h. After immunoprecipitation with α-Myc antibodies, the ubiquitination of OGT was analyzed by immunoblotting with α-HA antibodies. The same membrane was re-probed with α-Myc antibodies. Equal amounts of total lysates were subjected to immunoblotting with the indicated antibodies. e Expression vectors encoding Flag-XIAP WT and XIAP S406A were transfected into HCT116 cells. Cells were treated with 20 μM of etoposide for 24 h and lysed with NET buffer before being harvested. Activated caspase 3 was monitored with α-cleaved caspase 3 antibodies. Equal amounts of total lysates were subjected to immunoblotting with the indicated antibodies. f HCT116 cells were transfected with Myc-XIAP WT or Myc-XIAP S406A. Cells were treated with 20 μM of etoposide for 24 h and lysed with NET buffer before being harvested. Aliquots containing 50 μg of lysate were added to 100 μM of Ac-DEVD-AFC for measuring caspase-3/7 activity ( n = 6 per condition). Initial rates were analyzed at Ex/Em = 380 / 500 nm. β-actin or GAPDH was used as a loading control. Data are presented as means ± SD of at least three independent experiments. Statistical significance was determined using one-way analysis of variance. * P < 0.05, *** P < 0.001.