FIG 8.
Fusion of RCT domain of Rap1 to TIN2 alleles. (A) Schematic of the Rap1 RCT-domain TIN2 fusion protein indicating regions of shelterin binding (TRF1, TRF2, and TPP1) and location of mutations identified in DC patients. The RCT domain of mouse Rap1 (amino acids [aa] 295 to 393) was fused to the N terminus (aa 2) of TIN2 and L247E to generate RCT-TIN2 alleles. All alleles have an N-terminal FLAG-HA2 tag. The residue in red indicates the mutation introduced to generate the TRF1 binding mutant. (B) The indicated FLAG-HA2-tagged proteins were introduced into TIN2F/F MEFs. Immunoblotting for the indicated proteins was conducted in cell extracts from MEFs with or without Cre treatment (72 h). (C) Extracts from TIN2F/F MEFs infected with empty vector (EV) or the indicated FLAG-HA2 TIN2 alleles following Cre treatment (72 h) were immunoprecipitated (IP) with anti-HA resin, and immunocomplexes were probed for the indicated proteins. (D) Extracts from TIN2F/F MEFs infected with EV or the indicated FLAG-HA2 RCT-TIN2 alleles following Cre treatment (72 h) were immunoprecipitated with anti-HA resin, and immunocomplexes were probed for the indicated proteins.