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| 产品目录号 | GKC-LMP010071 | ||
| 产品名称 | Human GSK3A-A549 KO Cell Pool | ||
| 基因编号 | GSK3A | ||
| Uniprot_id | P49840 | ||
| 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 | Constitutively active protein kinase that acts as a negative regulator in the hormonal control of glucose homeostasis, Wnt signaling and regulation of transcription factors and microtubules, by phosphorylating and inactivating glycogen synthase (GYS1 or GYS2), CTNNB1/beta-catenin, APC and AXIN1 (PubMed:11749387, PubMed:17478001, PubMed:19366350). Requires primed phosphorylation of the majority of its substrates (PubMed:11749387, PubMed:17478001, PubMed:19366350). Contributes to insulin regulation of glycogen synthesis by phosphorylating and inhibiting GYS1 activity and hence glycogen synthesis (PubMed:11749387, PubMed:17478001, PubMed:19366350). Regulates glycogen metabolism in liver, but not in muscle (By similarity). May also mediate the development of insulin resistance by regulating activation of transcription factors (PubMed:10868943, PubMed:17478001). In Wnt signaling, regulates the level and transcriptional activity of nuclear CTNNB1/beta-catenin (PubMed:17229088). Facilitates amyloid precursor protein (APP) processing and the generation of APP-derived amyloid plaques found in Alzheimer disease (PubMed:12761548). May be involved in the regulation of replication in pancreatic beta-cells (By similarity). Is necessary for the establishment of neuronal polarity and axon outgrowth (By similarity). Through phosphorylation of the anti-apoptotic protein MCL1, may control cell apoptosis in response to growth factors deprivation (By similarity). Acts as a regulator of autophagy by mediating phosphorylation of KAT5/TIP60 under starvation conditions which activates KAT5/TIP60 acetyltransferase activity and promotes acetylation of key autophagy regulators, such as ULK1 and RUBCNL/Pacer (PubMed:30704899). Negatively regulates extrinsic apoptotic signaling pathway via death domain receptors. Promotes the formation of an anti-apoptotic complex, made of DDX3X, BRIC2 and GSK3B, at death receptors, including TNFRSF10B. The anti-apoptotic function is most effective with weak apoptotic signals and can be overcome by stronger stimulation (By similarity). Phosphorylates mTORC2 complex component RICTOR at 'Thr-1695' which facilitates FBXW7-mediated ubiquitination and subsequent degradation of RICTOR (PubMed:25897075). | ||
| 细胞系生成 | 采用CRISPR方法生成Human GSK3A-A549 KO Cell Pool | ||
| 数据说明 | Sanger 测序结果显示Human GSK3A-A549 KO Cell Pool敲除效率为100% | ||
| 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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