DNA constructs

The full-length rat 270 kDa AnkG-GFP has been described (Zhang and Bennett, 1998). The Flag-tagged full-length human AnkR (Flag-AnkR) was generated from the Ank1 isoform 1 ({"type":"entrez-nucleotide","attrs":{"text":"NM_020476.2","term_id":"70780358","term_text":"NM_020476.2"}}NM_020476.2 CDS) as described previously (Ho et al., 2014). The construct pEGFP-N1-AnkR was made with GeneArt Seamless Cloning and Assembly Kit (Thermo Fisher Scientific, cat. No. A13288). The primers for amplifying full-length AnkR from Flag-AnkR are forward: ATCTCGAGATGCCCTATTCTGTGG, and reverse: AGCTTGAGGGGGTTGGGTGTCGA. The primers for amplifying pEGFP-N1 are forward: CCAACCCCCTCAAGCTTCGAATTCTG, and reverse: TAGGGCATCTCGAGATCTGAGTCC. To generate pEGFP-N1-AnkR/G chimera, we first generate pEGFP-N1-AnkR (MBD-SBD)-AnkG (GE-270-RD) construct. pEGFP-N1-full length AnkR was cut with restriction enzymes EcoRI and Acl1 and ligated with two fragments, AnkR SBD C-terminal half and AnkG GE-270-RD, using GeneArt Seamless Cloning and Assembly Kit (Thermo Fisher Scientific, cat. No. A13288). The primers for amplifying AnkR SBD C-terminal half from pEGFP-N1-AnkR are forward: CCCCTGGTACAGGCAACGTTCCCGGAGAATG, and reverse: ACTGTTTTGTATCGCAGGGCCAG. The primers for amplifying AnkG GE270-RD from rat 270 kDa AnkG-GFP are forward: TGCGATACAAAACAGTTGAACGGAG, and reverse: GTACCGTCGACTGCAGAATTCGGTGGGCTTTCTTCTC. After generating pEGFP-N1-AnkR (MBD-SBD)-AnkG (GE-270-RD) construct, we constructed pEGFP-N1-AnkR (MBD-SBD)-AnkG (GE-270)-AnkR (RD) (i.e., AnkR/G chimera), by amplifying DNA fragment encoding AnkR (RD) by PCR using Flag-AnkR as template and introduced into the EcoRV-EcoRI sites of the pEGFP-N1-AnkR (MBD-SBD)-AnkG (GE-270-RD) construct. The primers for amplifying AnkR (RD) are forward: TCCGATATCAGCATTCTCAGTGAGTCC, and reverse: TAGAATTCGGGGGTTGGGTGTCGAGGTG.