Fig. 4

GNIP1 promotes the degradation of 14-3-3ζ by ubiquitination. A Interaction of Vps34 with 14-3-3ζ in H1299 cells. B Co-Immunoprecipitation assays of GNIP1 and 14-3-3ζ. C An exogenous interaction GNIP1 with 14-3-3ζ in H1299 cells. D Western blot was used to detect the degradation rate of 14-3-3ζ after CHX treatment in A549 cells. E 25 μg/ml CHX, 10 μM MG132 or 20 μM CQ were used to infect A549 cells for 12 h, and then performed western blot to detect the protein level of 14-3-3ζ (left panel). Quantitative statistical analysis of 14-3-3ζ protein (right panel). Data are presented as the average of three independent experiments (mean ± SD). *, p < 0.05, ***, p < 0.001, ****, p < 0.0001. F GNIP1 plasmids were transfected in A549 cells, the degradation rate of 14-3-3ζ was checked by western blot (left panel). Quantitative statistical degradation curve of 14-3-3ζ protein (right panel). Data are presented as the average of three independent experiments (mean ± SD). ***, p < 0.001. G Overexpressing GNIP1 in H1299, and treated with 10 μM MG132 for 12 h, the ubiquitination level of 14-3-3ζ was checked by western blot. H, I Overexpressing GNIP1 in H1299, and treated with 10 μM MG132 for 12 h, the K48-and K63-ubiquitination levels of 14-3-3ζ were analyzed by Western blot J Ectopic GNIP1 increases BECN1-VPS34 interactions. K H1299-LC3 stable cells were co-transfected with Flag-GNIP1 and Flag-14-3-3ζ plasmids. The expression of LC3B and P62 were detected by western blot. L The number of autophagosomes in H1299 cells after overexpression of GNIP1 or 14-3-3ζ was observed by fluorescence microscope