Fig. 3
Identification of binding proteins for ATF7IP’s C-terminal FNIII domain. a An experimental design for identification of binding proteins of ATF7IP’s FNIII domain by pull-down assay followed by LC–MS/MS analysis. b A list of top 20 proteins identified in the proteomic analysis. The proteins which was confirmed to interact with ATF7IP by independent experiment are filled in blue. ATF7IP is filled in red. c Co-IP assay performed with HEK293T cells transiently co-transfected with 3xFLAG-tagged ATF7IP WT and V5-tagged proteins. MBD1, a known ATF7IP interactor, was used as a positive control. All the high-ranked proteins bound to ATF7IP. d Co-IP assay performed with HEK29T cells transiently co-transfected with 3xFLAG-tagged ATF7IP dFNIII mutant and V5-tagged indicated proteins plus SETDB1. The FNIII mutant bound to SETDB1, but not to other proteins. e Co-IP assay performed with HEK29Tcells transiently co-transfected with 3xFLAG-tagged ATF7IP WT and V5-tagged ZMYM2 WT or mutants. FAM1 Mut possesses substitutions, V187R and L190R; FAM2 Mut possesses substitutions V217R and L220R; FAM1&FAM2 Mut possesses all the four substitutions; Δ39aa mutant lacks the region of 182–220 amino acids of ZMYM2. f FNIII domain of ATF7IP is sufficient for the interaction with ZMYM2. GST-pull down assay with recombinant FNIII domain of ATF7IP and cells lysates from HEK293T cells transiently transfected with V5-ZMYM2 WT or FAM1&2 Mut was performed. The pulled-down samples were analyzed by WB. g Alignment of MBD1-resembled sequences within the indicated proteins and their sequence logo. Asterisk indicates important residues for the interaction with ATF7IP. The substitutions of the I/V and L with R disrupted the interaction with ATF7IP. This consensus sequence is proposed as a FN III domain of ATF7IP-interacting Motif (FAM). The sequence logo was created by WEBLOGO (version 2.8.2)