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| 产品目录号 | GKC-LMP010076 | ||
| 产品名称 | Human ITCH-A549 KO Cell Pool | ||
| 基因编号 | ITCH | ||
| Uniprot_id | Q96J02 | ||
| 宿主细胞 | A549 | ||
| 组织来源 | 人非小细胞肺癌细胞 | ||
| 规格 | 1×106cells/T25培养瓶或1×106cells/冻存管 | ||
| 培养基 | MEM+10%FBS+1%P/S | ||
| 筛选标记 | N/A | ||
| 生长特性 | 贴壁细胞，上皮细胞样 | ||
| 培养条件 | 37℃，5% CO2的培养箱 | ||
| 传代比例 | 1/2到1/3传代，2-3天长满 | ||
| 筛选标记 | N/A | ||
| 换液频率 | 2-3天换液 | ||
| 支原体检测结果 | 阴性 | ||
| 蛋白组验证结果 | N/A | ||
| 抗体验证结果 | N/A | ||
| 目标基因介绍 | Acts as an E3 ubiquitin-protein ligase which accepts ubiquitin from an E2 ubiquitin-conjugating enzyme in the form of a thioester and then directly transfers the ubiquitin to targeted substrates (PubMed:14602072, PubMed:17028573, PubMed:16387660, PubMed:18718448, PubMed:18718449, PubMed:11046148, PubMed:19592251, PubMed:19116316, PubMed:19881509, PubMed:20491914, PubMed:20392206, PubMed:20068034, PubMed:23146885, PubMed:24790097, PubMed:25631046, PubMed:15051726). Catalyzes 'Lys-29'-, 'Lys-48'- and 'Lys-63'-linked ubiquitin conjugation (PubMed:17028573, PubMed:18718448, PubMed:19131965, PubMed:19881509). Involved in the control of inflammatory signaling pathways (PubMed:19131965). Essential component of a ubiquitin-editing protein complex, comprising also TNFAIP3, TAX1BP1 and RNF11, that ensures the transient nature of inflammatory signaling pathways (PubMed:19131965). Promotes the association of the complex after TNF stimulation (PubMed:19131965). Once the complex is formed, TNFAIP3 deubiquitinates 'Lys-63' polyubiquitin chains on RIPK1 and catalyzes the formation of 'Lys-48'-polyubiquitin chains (PubMed:19131965). This leads to RIPK1 proteasomal degradation and consequently termination of the TNF- or LPS-mediated activation of NFKB1 (PubMed:19131965). Ubiquitinates RIPK2 by 'Lys-63'-linked conjugation and influences NOD2-dependent signal transduction pathways (PubMed:19592251). Regulates the transcriptional activity of several transcription factors, and probably plays an important role in the regulation of immune response (PubMed:18718448, PubMed:20491914). Ubiquitinates NFE2 by 'Lys-63' linkages and is implicated in the control of the development of hematopoietic lineages (PubMed:18718448). Mediates JUN ubiquitination and degradation (By similarity). Mediates JUNB ubiquitination and degradation (PubMed:16387660). Critical regulator of type 2 helper T (Th2) cell cytokine production by inducing JUNB ubiquitination and degradation (By similarity). Involved in the negative regulation of MAVS-dependent cellular antiviral responses (PubMed:19881509). Ubiquitinates MAVS through 'Lys-48'-linked conjugation resulting in MAVS proteasomal degradation (PubMed:19881509). Following ligand stimulation, regulates sorting of Wnt receptor FZD4 to the degradative endocytic pathway probably by modulating PI42KA activity (PubMed:23146885). Ubiquitinates PI4K2A and negatively regulates its catalytic activity (PubMed:23146885). Ubiquitinates chemokine receptor CXCR4 and regulates sorting of CXCR4 to the degradative endocytic pathway following ligand stimulation by ubiquitinating endosomal sorting complex required for transport ESCRT-0 components HGS and STAM (PubMed:14602072, PubMed:23146885, PubMed:34927784). Targets DTX1 for lysosomal degradation and controls NOTCH1 degradation, in the absence of ligand, through 'Lys-29'-linked polyubiquitination (PubMed:17028573, PubMed:18628966, PubMed:23886940). Ubiquitinates SNX9 (PubMed:20491914). Ubiquitinates MAP3K7 through 'Lys-48'-linked conjugation (By similarity). Involved in the regulation of apoptosis and reactive oxygen species levels through the ubiquitination and proteasomal degradation of TXNIP (PubMed:20068034). Mediates the antiapoptotic activity of epidermal growth factor through the ubiquitination and proteasomal degradation of p15 BID (PubMed:20392206). Ubiquitinates BRAT1 and this ubiquitination is enhanced in the presence of NDFIP1 (PubMed:25631046). Inhibits the replication of influenza A virus (IAV) via ubiquitination of IAV matrix protein 1 (M1) through 'Lys-48'-linked conjugation resulting in M1 proteasomal degradation (PubMed:30328013). Ubiquitinates NEDD9/HEF1, resulting in proteasomal degradation of NEDD9/HEF1 (PubMed:15051726). | ||
| 细胞系生成 | 采用CRISPR方法生成Human ITCH-A549 KO Cell Pool | ||
| 数据说明 | Sanger 测序结果显示Human ITCH-A549 KO Cell Pool敲除效率为99% | ||
| 应用 | 体内和体外测定 | ||
| 复苏 | 1）在37℃水浴中预热完全培养基。 2）将冻存管在 37℃水浴中解冻1-2分钟。 3）将冻存管转移到生物安全柜中，并用70%乙醇擦拭表面。 4）拧开冻存管管盖，将细胞悬液轻轻转移到含有9mL完全培养基的无菌离心管中。 5）在室温下以125g离心5-7分钟，弃上清。 6）用5mL的完整培养基重悬细胞沉淀，将细胞悬液转移到T25培养瓶中。 7）将细胞转移到37℃，5% CO2的培养箱中培养。 8）参考传代比例：1/2到1/3传代，2-5天长满。 | ||
| 传代 | 1）待培养瓶中细胞汇合度至80%-90%以上，可进行细胞传代。 2）将培养基、PBS、胰酶（0.25%Trypsin_EDTA Gibco 25200-056）等从4℃冰箱中拿出，置于37℃水浴中温度接近37℃时取出并在瓶子表面喷洒75%酒精后置于生物安全柜中。 3）从培养箱中取出待传代的培养瓶，瓶身喷洒75%酒精后置于生物安全柜中。 4）为避免冲散细胞，沿培养瓶上壁PBS润洗细胞，清洗细胞后弃去，T25加2mL。 5）加入对应体积的胰酶（T75加1.5mL，T25加0.5mL），并轻轻晃动瓶身使胰酶平铺满细胞底部。可根据实际情况适当增加或减少用量。约1-2min后大部分细胞脱落时，加入对应体积的完全培养基终止消化，并用5mL移液管轻轻吹打至细胞全部脱落。 6）将细胞悬液转移至15mL离心管，悬液300g离心5min，弃上清。 7）移取5mL完全培养基重悬细胞，按需求调整接种比例，并补充培养瓶中完全培养基，T75加至13-15mL，T25加至5mL，加1%双抗。 8）盖上瓶盖拧紧后轻轻晃动瓶身，使细胞混合均匀后置于37℃，5% CO4培养箱中。 | ||
| 细胞冻存 | 1）准备冻存液，并提前预冷。 2）确保待冻存的细胞满足冻存要求，用显微镜检查以下状态：健康的外观及形态特征、所处生长周期（对数晚期）、无污染或衰退迹象。 3）对细胞进行消化及离心处理（具体步骤参考传代培养流程） 4）按照每管1mL的量添加冻存液重悬细胞，吹打均匀后分装至冻存管。 5）将细胞放在程序降温盒中，在-80℃冰箱中冷冻。 6）后续将细胞转移到液氮罐中，以便长期储存。 | ||
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