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. Author manuscript; available in PMC: 2024 Mar 16.
Published in final edited form as: Mol Cell. 2023 Mar 9;83(6):927–941.e8. doi: 10.1016/j.molcel.2023.02.023

Fig. 4. The interaction between the AHD3 domain of TNIP1 and the Zinc-Finger domain of TAX1BP1 is necessary for TNIP1’s mitophagy inhibition.

Fig. 4

(A) Left, High Confidence Candidate Interaction Proteins (HCIPs) network of HA-TNIP1 following an HA-pull down and mass spectrometry analysis. The size of the bait represents higher Z score interaction. Right, heat map of the interactors with wild type TNIP1 or ΔAHD3 and ΔAHD4 mutants.

(B) Co-IP and IB of HEK293T cells stably expressing HA-TNIP1 full length, ΔAHD1, ΔAHD3 and ΔAHD4 constructs or HA-TNIP3 using magnetic HA beads.

(C) Top, schematic representation of full length TAX1BP1 and the regions encompassing the coiled-coil domain (CC), SKICH truncation (ΔSKICH) and Zinc-Finger truncation (ΔZinc-Finger) constructs. Bottom, Co-IP and IB of HEK293T cells stably expressing GFP-TNIP1 full length and transiently expressing HA-TAX1BP1 full length, CC, ΔSKITCH and ΔZinc-Finger constructs using magnetic GFP beads.

(D) HeLa WT cells or TAX1BP1 KO stably expressing mito-mKeima, HA-Parkin and overexpressing FKBP-GFP-TNIP1 construct were treated with O/A for 3 h and subjected to FACS analysis. Left, representative FACS plot. Right, bar graph representing data as mean ± SEM obtained from 3 independent replicates.

(E) Co-IP and IB of HeLa TAX1BP1 KO cells stably expressing HA-TNIP1 full length or the ΔAHD1, ΔAHD3, ΔAHD4 constructs using magnetic HA beads. Ø: no overexpressed construct. * Marks unspecific band.