Fig. 5: MDM2 regulates ubiquitination of KL by interacting with KL.

A Left: Co-immunoprecipitation of MDM2 with KL. Lysates from A549 cells transfected with MDM2-HA and Flag-KL constructs were immunoprecipitated with anti-Flag antibodies. Immunoblotting analysis was performed to detect immunoprecipitated proteins. Right: Lysates from A549 cells transfected with MDM2-HA and Flag-KL constructs were immunoprecipitated with anti-HA antibodies. Immunoblotting analysis was performed to detect immunoprecipitated proteins. B Subcellular co-localization of endogenous KL and MDM2 in A549 cells visualized by confocal microscopy. Staining was carried out with rabbit anti-KL (green) and mouse anti-MDM2 (red) antibodies. C Endogenous KL associates with MDM2. Rat lung lysates were subjected to immunoprecipitation with mouse anti-MDM2 or rabbit anti-KL antibodies. D Post-nuclear supernatants of A549 cells were fractioned on an isopycnic 15–55% (w/w) linear sucrose gradient, and equal aliquots of the final equilibrium gradient fractions were immunoblotted using antibodies against GM130, calnexin and KL, Shank1 and MDM2. E A549 cells were transfected with vector or MDM2 constructs. Next, they were treated with/without MG132 and lysed. The protein level of KL was detected by western blotting. F The relative protein levels in (E) were shown (relative to vector expression level, the error bars indicate SEM for three experiments, **p < 0.01, determined by student’s t test). G A549 cells were transfected with either vector (pCDNA3.1), pCDNA3.1-MDM2, or si-NC, si-MDM2 for 48 h, and lysed by TNE buffer. The ubiquitination of KL was detected by IP and western blotting with Ub antibody. H The relative Ub levels in (G) are shown (relative to vector or siNC expression level, the error bars indicate SEM for three experiments, **p < 0.01, determined by student’s t test).