客户采用我司链霉亲和素磁珠进行RNA pull down实验

RNA pull-down and RNA immunoprecipitation (RIP) assays
The biotinylated probes against linear MYBL2 and the junction site of circ-MYBL2 were designed and synthesized by Sangon (Shanghai, China). Then, the probes were added into MM cell lysates and incubated overnight. After washing, PuriMag G-streptavidin (PuriMag G Series, Xiamen, China) was added into above lysates, and incubated for 3 h. The complexes enriched by these probes were eluted for qRTPCR or mass spectrum/western blot analysis. For RIP assay, the EZ-Magna RIP™ RNA-Binding Protein IP Kit (Millipore, Schwalbach, Germany) with 5 μg anti-Cyclin F was used as per manufacturer’s protocol.

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更多原文见如下：

circRNA circ-MYBL2 is a novel tumor suppressor and potential biomarker in multiple myeloma

Human Cell volume 34, pages219–228(2021)

ABSTRACT：Currently, multiple myeloma (MM) is still an incurable disease. Deciphering its pathogenesis will bring new targets for clinical diagnosis and treatment. In the present study, we identified a MM-associated circular RNA (circRNA), circ-MYBL2, which was dramatically decreased in MM tissue and serum samples in comparison to normal samples. Low circ-MYBL2 level was closely correlated with high clinical stage and unfavorable outcome, and serum circ-MYBL2 had excellent accuracy in diagnosing MM. Exogenous circ-MYBL2 expression notably repressed MM cell viability, DNA synthesis and cell cycle progression. Further exploration revealed that circ-MYBL2 exerted the tumor-inhibiting effect by affecting the phosphorylation level of its linear isoform, in which circ-MYBL2 facilitated the binding of Cyclin F to MYBL2, dampening MYBL2 phosphorylation and activation, thereby inhibiting the transcription of a number of well-known proliferation-related oncogenes. Importantly, overexpression of circ-MYBL2 significantly reduced the tumor size of subcutaneous xenografts in nude mice. Taken together, our data unveil a regulatory mechanism linking circ-MYBL2 and its host gene mediated by Cyclin F, providing a potential diagnostic, prognostic and therapeutic target for MM patients.

当前，多发性骨髓瘤（MM）仍是不治之症。破译其发病机理将为临床诊断和治疗带来新的靶点。在本研究中，我们确定了与MM相关的环状RNA（circRNA），circ-MYBL2，与正常样品相比，其在MM组织和血清样品中显着减少。 circ-MYBL2水平低与临床分期高，预后不良有关，血清circ-MYBL2在MM诊断中具有极好的准确性。外源性circ-MYBL2表达显着抑制了MM细胞的活力，DNA合成和细胞周期进程。进一步的研究表明，circ-MYBL2通过影响其线性同工型的磷酸化水平来发挥抑瘤作用，其中circ-MYBL2促进Cyclin F与MYBL2的结合，抑制MYBL2的磷酸化和活化，从而抑制了许多转录。增殖相关癌基因的鉴定重要的是，circ-MYBL2的过表达显着降低了裸鼠皮下异种移植物的肿瘤大小。综上所述，我们的数据揭示了一种连接circ-MYBL2及其细胞周期蛋白F介导的宿主基因的调控机制，为MM患者提供了潜在的诊断，预后和治疗靶标。