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. 2015 Sep 30;6:8419. doi: 10.1038/ncomms9419

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(a) Engineer of TAP-KLF4 stable clone and purification of the KLF4 protein complex. Proteins that interacted with KLF4 were purified from HeLaS3 cells stably expressing Flag and HA-tagged KLF4 or HeLaS3 (control). The nuclear extract was used for purification of KLF4 complex following by the standard TAP purification protocol. (b) Validation of PRMT5-KLF4 interaction in vivo. Endogenous interaction between KLF4 and PRMT5 was observed in HeLaS3 cell measured by immunoprecipitation. Normal IgG was used as the control. (c) Confirmation of interaction between KLF4 and PRMT5 using immunoprecipitation based on the TAP-KLF4 stable clone utilized for the purification of KLF4 complex in (a). (d,e) Validation of PRMT5-KLF4 interaction by immunoprecipitation based on expression and immunoprecipitation of KLF4 with different tag (HA). Cell lysates from HEK293T cells ectopically expressed the indicated plasmids were immunoprecipitated with the antibody again HA tag (IP: HA-KLF4/IB: PRMT5) or PRMT5 (IP: HA-PRMT5/IB: Flag-KLF4). (f) Direct interaction between PRMT5 and KLF4. Cell lysates from HEK293T cells transfected with HA-tagged PRMT5 were mixed with purified GST or GST-KLF4 immobilized on glutathione beads. Samples were electrophoresed and immuoblotted with the HA tag antibody. Levels of input protein are shown by immunoblotting with the HA tag antibody or Coomassie staining of SDS-PAGE gel.