His-Ubiquitin Pull-Down Assay

Full-length WISP1 was cloned into pCMV-Tag 2B vector, and mutations were introduced into WISP1 with QuikChange II Site-directed Mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA). The generated plasmids were designated as Flag-WISP1 (WT), Flag-WISP1 (K50R), Flag-WISP1 (K190R), and Flag-WISP1 (K268R). RNF180 sequence containing a myc tag was obtained from GENEWIZ, lnc. (Suzhou, China) and further cloned into p-DONR221 vector for expression. Human ubiquitin with 6xHis tag was cloned into pcDNA-DEST40. All DNA constructs were sequence verified. Wild-type WISP1 (WT) or WISP1 mutants were transfected into HEK293T cells, along with myc-RNF180 and His-Ub constructs using Lipofectamine 2000 reagent according to manufacturer's instructions. 48 h later, cells were lysed and incubated with Ni-NTA agarose beads (Qiagen, Hilden, Germany). The complexes were washed, eluted, and applied onto SDS-PAGE gels for immunoblotting.