TAX1BP1 ^CC2 and FIP200 ^Claw are sufficient to interact with NBR1 in vivo. (A) Representative confocal micrographs of HeLa PentaKO cells co-transduced with tf-NBR1 and indicated BFP-TAX1BP1 variants. Scale bar: 10 µm. (B) Quantification of A. Data represent median of three independently generated cell lines. Pearson’s correlation coefficient between RFP and BFP (reflective of NBR1 and TAX1BP1 co-localization) was calculated using Cell Profiler. Scatter plots represent median ± SD. Each data point represents a single replicate. n ≥ 17 cells per replicate. P values were determined using one-way ANOVA (P < 0.0001). Multiple comparisons were made using Tukey correction. ****, P < 0.0001; ns, not significant. (C) Representative confocal micrographs of HeLa PentaKO cells co-transduced with tf-NBR1 and BFP-FIP200^Claw. Scale bar: 10 µm. (D) Quantification of C. Data represent median of three independently generated cell lines. Pearson’s correlation coefficient between RFP and BFP (reflective of NBR1 and FIP200^Claw co-localization) was calculated using Cell Profiler. Scatter plots represent median ± SD. Each data point represents a single replicate. n > 16 cells per replicate. P values were determined using one-way ANOVA (P < 0.0001). Multiple comparisons were made using Tukey correction. ****, P < 0.0001. (E) K_D values (±standard error of the fit) for NBR1^722–745, NBR1^725–749, and GST-His₆ control. Values are derived from ForteBio Octet BLI analysis using purified GST-His-tagged peptides immobilized on NiNTA sensors and probed against titrations of MBP-LC3A, MBP-FIP200^Claw, TRX-TAX1BP1^CC2-MBP, and MBP-GABARAPL1. Experiments were performed in duplicate (separate days). n.b., no detectable binding.