Generation of HA-tagged Fusion Proteins

To generate HA-tagged fusion proteins, each DNA fragment encoding ANK1-F1 (aa 1–318), ANK1-F2 (aa 250–568), ANK1-F3 (aa 500–827), or the last three extracellular loops of band 3 (band 3-L4, L5, L6) containing a HA tag at the C terminal was amplified from the cDNA (reversely transcribed by the human reticulocyte mRNA) using six respective reverse primers which introduced the HA tag nucleotide sequence and six respective forward primers listed in Table S3. PCR products were cloned into the BamHI and XhoI sites of pET30a or pGEX-6P-1 vector. The pET30a plasmids carrying ANK1-F1, -F2, -F3 gene fragments and the pGEX-6P-1 plasmids carrying band 3-L4, L5, L6 gene fragments were transformed into E. coli BL21 (DE3) pLysS cells for expression. Protein expression was induced with 0.5 mM IPTG for 7 h at 37 °C. Soluble proteins were purified by Talen Biotech as described previously (62 (link)). Additionally, the buffer of eluted proteins was exchanged to PBS using 10 kDa Amicon spin columns (Millipore).