Figure 1

Construction of Eno2 mutant protein expression plasmid. A two-step PCR method was used to generate the expression plasmid, with the mutation of aspartic acid (D) to serine (S) at position 419 (Eno2-[D419S]) as an example. Four primers (p1 to p4) were used for the PCR, where p1 and p4 represent the forward and reverse flanking primers of the eno2 fragment, and p2 and p3 are complementary mutagenic primers carrying the mutation sequence. The plasmid pCS2-Eno2-wb- Flag containing the mouse eno2 gene fused with a Flag reporter gene and with wobble-modified nucleotides (wb) was used as the template in the first PCR step. Primers p1 and p2 were used to produce the upstream fragment of the gene containing the mutation site (X), while p3 and p4 were used to produce the downstream fragment. In the second PCR step, the products from the first step were mixed, and a small amount of p1 and p4 primers were added to generate the complete mutant gene fragment. After PCR completion, the mutant gene fragment and the pCS2 plasmid were digested with EcoRI and XhoI restriction enzymes to produce sticky-ended inserts and vectors. Finally, the insert and vector were ligated to create the pCS2-derived expression plasmid for the Eno2 mutant protein fused with the Flag reporter gene. Blue color: eno2 cDNA; orange color: reporter Flag; red color: mutation site; grey color: plasmid pCS2⁺ backbone.