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. 2020 Mar 31;8:208. doi: 10.3389/fcell.2020.00208

FIGURE 2.

FIGURE 2

Variants of LC3B and GATE-16 are selective to their cognate LIR and have high affinity in vitro. (A) Phage ELISA of LC3Bwt and GATE-16wt and selected variants with specificity to the LIR motifs of OPTN, p62 or NDP52. Binding of OPTN.LC3Bv and p62.GATE-16v was tested against ATG3, ATG4B, p62, OPTN, NIX and BNIP3. Variant NDP52.LC3Bv was tested against p62, OPTN, BNIP3, NIX, NDP52 and NPD52 ΔLIR. Background correction to BSA signal was applied. (B,C) Sequences of the LC3B and GATE-16 variants with binding to OPTN, NDP52 and p62. Dashes indicate wt residues. Color coding of region 1 and region 2 as in Figure 1C. (D–G) Biolayer interferometry (BLI) measurements of LC3Bwt, GATE-16wt (upper panels) and engineered variants (lower panels) against their cognate LIR-peptide. (C) LC3Bwt and OPTN.LC3Bv binding to OPTN peptide. (D) LC3Bwt and OPTN.LC3Bv binding to pS177 OPTN peptide. (E) LC3Bwt and NDP52.LC3Bv binding to NDP52 LIR peptide. (F) GATE-16wt and p62.GATE-16v binding to p62 LIR peptide.