TNIP1 interacts with human ATG8 family proteins through a LIR motif. (A) Schematic drawing of the domain architecture of TNIP1, showing possible LIRs. (B) Peptide array of 20-mer peptides covering full length TNIP1 was used to probe for possible LIRs, using GST-GABARAP. (C) Amino-acid sequence alignment showing conservation of the core consenesus LIRs in TNIP1 across species. TNIP1 amino acid sequences were collected from UniProt, and multiple sequence alignment performed with Clustal Omega. Asterisk (*) indicates fully conserved residues; colon (:) indicates conservation between groups of strongly similar properties (>0.5 Gonnet PAM 250 matrix); and a period (.) indicates conservation between groups of weakly similar properties (0–0.5 Gonnet PAM 250 matrix). Mutated residues for the LIR mutants (mLIR1 and mLIR2) are shown in red. (D) In vitro GST-pulldown assay using 35S-labeled myc-TNIP1, myc-TNIP1-F83A/L86A (mLIR1), myc-TNIP1-F125A/V128A (mLIR2) and myc-TNIP1-F83A/L86A/F125A/V128A (mLIR1+2) against recombinant GST and GST-tagged human ATG8s. Bound myc-TNIP1 WT and LIR mutants were detected by autoradiography (AR). (E) Quantification of GST-pulldown from D. Relative % binding was quantified against 10% input (n = 3). * = P < 0.05, ** = P < 0.01, based on one-way ANOVA (post hoc Tukey test). Error bars indicate SEM. (F) LIR mutation impacts in vivo interaction with LC3A/B. Anti-HA AP of cells expressing HA-TNIP1^WT and HA-TNIP1^mLIR1+2 were performed followed by Western blot against indicated proteins. LIR mutation reduced the interaction between TNIP1 and LC3A/B. (G) Quantification of blots exemplified in panel F (n = 3). Error bars indicate SEM. ** = P < 0.01, unpaired, two-sided t test. In E and G, data distribution was assumed to be normal, but this was not formally tested. Source data are available for this figure: SourceData F4.