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客戶采用我司鏈霉親和素磁珠進(jìn)行RNA pull down實(shí)驗(yàn)

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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更多原文見如下：

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.

當(dāng)前，多發(fā)性骨髓瘤（MM）仍是不治之癥。破譯其發(fā)病機(jī)理將為臨床診斷和治療帶來新的靶點(diǎn)。在本研究中，我們確定了與MM相關(guān)的環(huán)狀RNA（circRNA），circ-MYBL2，與正常樣品相比，其在MM組織和血清樣品中顯著減少。 circ-MYBL2水平低與臨床分期高，預(yù)后不良有關(guān)，血清circ-MYBL2在MM診斷中具有極好的準(zhǔn)確性。外源性circ-MYBL2表達(dá)顯著抑制了MM細(xì)胞的活力，DNA合成和細(xì)胞周期進(jìn)程。進(jìn)一步的研究表明，circ-MYBL2通過影響其線性同工型的磷酸化水平來發(fā)揮抑瘤作用，其中circ-MYBL2促進(jìn)Cyclin F與MYBL2的結(jié)合，抑制MYBL2的磷酸化和活化，從而抑制了許多轉(zhuǎn)錄。增殖相關(guān)癌基因的鑒定重要的是，circ-MYBL2的過表達(dá)顯著降低了裸鼠皮下異種移植物的腫瘤大小。綜上所述，我們的數(shù)據(jù)揭示了一種連接circ-MYBL2及其細(xì)胞周期蛋白F介導(dǎo)的宿主基因的調(diào)控機(jī)制，為MM患者提供了潛在的診斷，預(yù)后和治療靶標(biāo)。