Fig. 4: USP24 interacts with Beclin1 and increases the stability of Beclin1 by decreasing its K48-linked ubiquitination.

A, B Reciprocal Co-IP experiments were performed to detect the interaction between USP24 and Beclin1. An IgG antibody was used as the control. C The cell double immunofluorescence assay was carried out using anti-USP24 and anti-Beclin1 antibodies. The colocalization of USP24 (red) and Beclin1 (green) was observed under a confocal microscope (n = 3). Scale bar, 10 μm. D The colocalization was evaluated by Mander’s colocalization coefficients in ten regions of interest (n = 10). E SMMC-7721 cells were treated with 10 μg/ml MG132 before harvesting. An anti-Beclin1 antibody was used to immunoprecipitate the Beclin1 protein, then an anti-Ubi antibody was used for western blot. F, G The levels of K48 and K63-linked ubiquitination were measured by western blot. H–K Control and USP24 overexpressed HCCLM3 and SMMC-7721 cells were treated with CHX (100 μg/ml) for indicated time. The expression level of Beclin1 was determined by western blot (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.