Anti flag antibody d6w5b

A mutational library was generated by substituting single amino acid changes in the LA3 domain of LDLR protein. Amino acids essential for maintaining the three-dimensional structure of the protein (i.e., cysteines that form disulfide bond, the amino acids that coordinate the calcium ion, and those forming the hydrophobic core) were not changed^{48 (link)}. Other amino acids in LA3 domain were mutated according to BLOSUM scoring matrix^{38 (link)}. The mutants were synthesized and cloned into the lentivirus vector pLV-EF1a-IRES-Hygro (Addgene, 85134) between the BamHI and MluI restriction enzyme sites (Genescript LLC). An N-terminal FLAG tag was added to each LA3 mutant to monitor expression. THP-1 cells were transduced to express each individual LA3 mutant and then inoculated with SINV-EEEV expressing GFP at an MOI of 10 for 20 h. Cells were also stained with an anti-FLAG antibody (D6W5B, Cell Signaling, USA) to measure the expression level of these mutants on cell surface.