数据库共享平台 -- 细胞查询-- 详细信息
       
产品目录号  GKC-LMP010045
产品名称  Human CUL4B-A549 KO Cell Pool
基因编号   CUL4B
Uniprot_id  Q13620
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   Core component of multiple cullin-RING-based E3 ubiquitin-protein ligase complexes which mediate the ubiquitination and subsequent proteasomal degradation of target proteins (PubMed:14578910, PubMed:16322693, PubMed:16678110, PubMed:18593899, PubMed:29779948, PubMed:30166453, PubMed:33854232, PubMed:33854239, PubMed:22118460). The functional specificity of the E3 ubiquitin-protein ligase complex depends on the variable substrate recognition subunit (PubMed:14578910, PubMed:16678110, PubMed:18593899, PubMed:29779948, PubMed:22118460). CUL4B may act within the complex as a scaffold protein, contributing to catalysis through positioning of the substrate and the ubiquitin-conjugating enzyme (PubMed:14578910, PubMed:16678110, PubMed:18593899, PubMed:22118460). Plays a role as part of the E3 ubiquitin-protein ligase complex in polyubiquitination of CDT1, histone H2A, histone H3 and histone H4 in response to radiation-induced DNA damage (PubMed:14578910, PubMed:16678110, PubMed:18593899). Targeted to UV damaged chromatin by DDB2 and may be important for DNA repair and DNA replication (PubMed:16678110). A number of DCX complexes (containing either TRPC4AP or DCAF12 as substrate-recognition component) are part of the DesCEND (destruction via C-end degrons) pathway, which recognizes a C-degron located at the extreme C terminus of target proteins, leading to their ubiquitination and degradation (PubMed:29779948). The DCX(AMBRA1) complex is a master regulator of the transition from G1 to S cell phase by mediating ubiquitination of phosphorylated cyclin-D (CCND1, CCND2 and CCND3) (PubMed:33854232, PubMed:33854239). The DCX(AMBRA1) complex also acts as a regulator of Cul5-RING (CRL5) E3 ubiquitin-protein ligase complexes by mediating ubiquitination and degradation of Elongin-C (ELOC) component of CRL5 complexes (PubMed:30166453). Required for ubiquitination of cyclin E (CCNE1 or CCNE2), and consequently, normal G1 cell cycle progression (PubMed:16322693, PubMed:19801544). Regulates the mammalian target-of-rapamycin (mTOR) pathway involved in control of cell growth, size and metabolism (PubMed:18235224). Specific CUL4B regulation of the mTORC1-mediated pathway is dependent upon 26S proteasome function and requires interaction between CUL4B and MLST8 (PubMed:18235224). With CUL4A, contributes to ribosome biogenesis (PubMed:26711351).
细胞系生成   采用CRISPR方法生成Human CUL4B-A549 KO Cell Pool
数据说明   Sanger 测序结果显示Human CUL4B-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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