TAX1BP1 is insensitive to negative charge and phosphorylation of −2, −1 LIR positions. (A) Phosphorylated peptide variants were generated for each LIR peptide containing phosphorylatable residues (Ser or Thr) at the −2 or −1 positions relative to the core LIR motif. The phosphorylated LIR peptide array was probed with 100 nM GST-LC3A, GST-GABARAPL1, GST-FIP200^Claw, or GST-TAX1BP1^CC2. Antibody-HRP conjugates were used to visualize the arrays. Peptide spots that bound none of the four protein probes (“never-binders”) are labeled with red asterisk and excluded from further analysis. (B) Spearman r correlation test was performed between phosphomimetic (Pm) probe in Fig. 4 A and phosphorylated (Ph) probe in A. “Never-binders” from phosphomimetic array were excluded from analysis. (C) Native, phosphomimetic, and phosphorylated peptide arrays were probed in parallel with TAX1BP1^CC2-MBP to allow direct comparison. (D) Comparison of spot intensity between native, phosphomimetic (Pm), and phosphorylated (Ph) variant peptides using nonparametric Friedman test one-way ANOVA with Dunn’s multiple comparison test. ns, not significant. (E) Spearman r correlation test was performed between TAX1BP1 phosphomimetic (Pm) probe in (Fig. 4 A) and technical replicate (C), and between TAX1BP1 phosphorylated (Ph) probe in (A) and technical replicate (C).