Fig. 5: LC3 directly associates with ELKS1 in-vitro and in-vivo. | Nature Communications

Fig. 5: LC3 directly associates with ELKS1 in-vitro and in-vivo.

From: Autophagy lipidation machinery regulates axonal microtubule dynamics but is dispensable for survival of mammalian neurons

Fig. 5: LC3 directly associates with ELKS1 in-vitro and in-vivo.The alternative text for this image may have been generated using AI.

a Mass spectrometry analysis of LC3 interaction partners in the brain. Volcano plot illustrates the significantly abundant proteins. The −log10 is plotted against the log2 (fold change: LC3/control). The non-axial vertical lines denote ±2-fold change while the non-axial horizontal line denotes p = 0.05, which is the significance threshold. b–d Ectopically expressed ELKS1, tagged with tdTomato is co-immunoprecipitated (Co-IP) with eGFP-tagged LC3A (b) and LC3B (c), but not with GABARAP (d). HEK cell lysates were directly analyzed (Input) or subjected to GFP immunoprecipitation (Elution) and further immunoblotted against mCherry (Co-IP). Input, 1% of the total lysate. e–g Ectopically expressed ELKS1, tagged with tdTomato is co-immunoprecipitated (Co-IP) with eGFP-tagged LC3A G120A (e) and LC3B G120A (f) but not with GABARAP G116A (g). HEK cell lysates were directly analyzed (Input) or subjected to GFP immunoprecipitation (Elution) and further immunoblotted against mCherry (Co-IP). Input, 1% of the total lysate. h, i Ectopically expressed LC3A and LC3B, tagged with eGFP are co-immunoprecipitated with tdTomato-ELKS1. HEK cell lysates were directly analyzed (Input) or subjected to ELKS1 immunoprecipitation (Elution) and further immunoblotted against GFP (Co-IP). Input, 1% of the total lysate. j Purified recombinant His6-LC3B, detected by immunoblotting with LC3B antibody, directly binds Myc-ELKS1. Input, 12.5% of the total recombinant LC3B and Myc-ELKS1. k Co-immunoprecipitation of endogenous ELKS1 with LC3B form mouse brain lysate. Input, 4% of the total lysate was added to the assay. l Co-immunoprecipitation of endogenous LC3B with ELKS1 form mouse brain lysate. Input, 4% of lysate. m, n Close colocalization of tdTomato-ELKS1 and eGFP-LC3B at en passant presynaptic boutons marked by synapsin immunofluorescence (blue). Colocalization is analyzed by Mander´s overlap coefficient (80.42 ± 7.22%). Scale bars: 5 µm (upper panel), 1 µm (lower panels). N = 2 independent experiments. All graphs show mean ± SEM. Co-IP experiments in HEK cells represent examples from N = 3 independent experiments. Source data are provided as a Source Data file.

Back to article page