NBR1 binds LC3A, FIP200 ^Claw , and TAX1BP1 ^CC2 through a shared unstructured region. (A) Schematic of NBR1 domain architecture. Domain boundaries are listed above the schematic. Truncation boundaries are listed below the schematic. (B) HEK293T cells were co-transfected with full-length (FL) myc-TAX1BP1 and the indicated BFP-V5-NBR1 truncations. Extracts derived from transfected cells were immunoprecipitated (IP) with protein G dynabeads conjugated with anti-BFP antibody. Input and eluates were resolved by SDS-PAGE followed by immunoblotting (IB) with the indicated antibodies. (C and D) HEK293T cells were transfected with indicated BFP-V5-NBR1 truncations. Extracts derived from transfected cells were incubated with GST-resin pre-bound with purified GST-MAP1LC3A or GST-FIP200^Claw. Input and eluates were resolved by SDS-PAGE followed by immunoblotting (IB) with the indicated antibodies. (E) Purified GST-MAP1LC3A, GST-FIP200^Claw, or GST-TAX1BP1^CC2 were serially diluted and incubated with 50 nM FITC-labeled NBR1^725–749. Fluorescence anisotropy was measured by plate reader and analyzed using a nonlinear fit to determine K_D. (F) Schematic of TAX1BP1 architecture. Domain boundaries are listed above the schematic. Truncation boundaries are listed below the schematic. Source data are available for this figure: SourceData FS1.