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. 2016 Mar 11;7:10965. doi: 10.1038/ncomms10965

Figure 2. TLX interacts with the miRNA processing machinery.

Figure 2

(a) A scheme for identifying TLX-interacting proteins using mass spectrometry (MS) analysis. (b) Differentially represented proteins in the HA immunoprecipitates of control HA or HA-TLX-expressing HeLa cells. Arrow indicates a protein band of 68 kD that is specifically detected in the HA immunoprecipitates of HA-TLX-expressing HeLa cells. (c) Interaction of TLX with p68, Drosha and DGCR8. Lysates of HA-TLX transfected HEK293T cells were treated with or without DNase and RNase, then immunoprecipitated with HA antibody or IgG control. The immunoprecipitates were blotted with p68 antibody. In parallel, lysates of Flag-Drosha and HA-TLX or Flag-DGCR8 and HA-TLX co-transfected HEK293T cells were treated with or without DNase and RNase. Cell lysates were immunoprecipitated with anti-Flag antibody, then blotted with anti-HA antibody. (d) Interaction of TLX with Drosha and DGCR8 in mouse brains. Lysates of embryonic mouse brains were immunoprecipitated with TLX antibody, then blotted with anti-Drosha, anti-DGCR8 or anti-TLX antibody. (e) A scheme for RNA immunoprecipitation. Lysates of NSCs transduced with TLX siRNA were immunoprecipitated with anti-Drosha, anti-DGCR8 or anti-TLX antibody. RNAs were extracted from the immunoprecipitates, and subjected to RT–PCR for pri-miR-219. (f) TLX knockdown promoted the binding of Drosha and DGCR8 to pri-miR-219. Lysates of NSCs transduced with siC or siTLX were immunoprecipitated with IgG control or indicated antibodies. pri-miR-219 RNA associated with Drosha (indicated by solid arrows) or DGCR8 (indicated by open arrows) was determined by RT–PCR.