Fig. 5

The ubiquitination of 14-3-3 ζ relies on the E3 ligase activity of GNIP1 A GNIP1or GNIP1-W57A plasmids were transfected in A549 cells, the degradation rate of 14-3-3ζ was checked after CHX treatment 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, ****, p < 0.0001, ns, no significance. B 14-3-3ζ plasmids and GNIP1or GNIP1-W57A plasmids were transfected in H1299 cells, and treated with 10 μM MG132 for 12 h, the ubiquitination level of 14-3-3ζ was checked by western blot. C, D Overexpressing 14-3-3ζ plasmids and GNIP1or GNIP1-W57A plasmids 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. E Interaction of GNIP1-W57A with GFP-LC3B in H1299 cells. F Interaction of GNIP1-W57A with HA-BECN1 in H1299 cells. G, H GNIP1 or GNIP1-W57A plasmids were transfected in H1299 and A549 cells, the cell growth ability was detected. Data are presented as the average of three independent experiments (mean ± SD). ****, p < 0.0001. I, J The scratch wound healing assays of H1299 and A549 cells with exogenous GNIP1 or GNIP1-W57A expression (upper panel). Statistical analysis of cell wound healing assays between groups (lower panel). Data are presented as the average of three independent experiments (mean ± SD). *, p < 0.05. **, p < 0.01, ***, p < 0.001. K, L Transwell assays of H1299 and A549 cells with exogenous GNIP1 or GNIP1-W57A expression