Site-directed mutagenesis (SDM)

PCR-based site-directed mutagenesis was carried out using the QuikChange^TM lightning site-directed mutagenesis kit (Agilent Technologies; Catalog No. 210518), following the company's protocol. Briefly, amino acid exchanges K16R were generated by point mutations in the pCl-neo-HA-Sp1 constructs. The following complementary primers, forward primer: 5′-GCTGTGGTGAGGATTGAAAAAGGAGTTGGTGGC-3′ and reverse primer: 5’-GCCACCAACTCCTTTTTAAATCCTCACCACAGC-3’ were used (changed nucleotides are in boldface type and underlined).

Epicurean Coli XL1-Blue super-competent cells (Stratagene) were transformed with resultant plasmid. The plasmid was amplified, and the mutation was confirmed by sequencing as described previously [101].