Immunoprecipitation of miRNP with anti-Ago1 (Abcam, Cambridge, MA) or anti-IgG (Sigma) was performed as previously described60 (link). In brief, cells were lysed in buffer containing 100 mM KCl, 5 mM MgCl2, 10 mM HEPES (pH7.4) and 0.5% NP-40; and the immune complex captured by protein A agarose was washed in buffer containing 150 mM KCl, 5 mM MgCl2, 10 mM HEPES (pH7.4) and 0.1% NP-40 for 6 times. RNA extraction was performed using RNAeasy Kit (Qiagen, Valencia, CA).
Cai J., Fang L., Huang Y., Li R., Xu X., Hu Z., Zhang L., Yang Y., Zhu X., Zhang H., Wu J., Huang Y., Li J., Zeng M., Song E., He Y., Zhang L, & Li M. (2017). Simultaneous overactivation of Wnt/β-catenin and TGFβ signalling by miR-128-3p confers chemoresistance-associated metastasis in NSCLC. Nature Communications, 8, 15870.
Other organizations :
Sun Yat-sen University, First Affiliated Hospital of Guangzhou Medical University, State Key Laboratory of Respiratory Disease, Guangzhou Medical University, Sun Yat-sen University Cancer Center, The First Affiliated Hospital, Sun Yat-sen University, Sun Yat-sen Memorial Hospital, Augusta University Health
Immunoprecipitation with anti-Ago1 antibody (Abcam, Cambridge, MA)
Immunoprecipitation with anti-IgG antibody (Sigma)
dependent variables
Captured immune complex
Extracted RNA
control variables
Cell lysis buffer containing 100 mM KCl, 5 mM MgCl2, 10 mM HEPES (pH7.4) and 0.5% NP-40
Wash buffer containing 150 mM KCl, 5 mM MgCl2, 10 mM HEPES (pH7.4) and 0.1% NP-40
RNAeasy Kit (Qiagen, Valencia, CA) for RNA extraction
controls
Positive control: Immunoprecipitation with anti-Ago1 antibody
Negative control: Immunoprecipitation with anti-IgG antibody
Annotations
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