FIGURE 3.

SKP2 enhances the K48‐linked poly‐ubiquitination of Beclin1. (A) The expression pattern of SKP2 in microarray data set GSE110711 of mice with silicosis in GEO database, with SiO₂ representing silicosis model and UT representing the control. (B) IHC analysis of SKP2 in alveolar tissues of mice treated with CS or saline. C, RT‐qPCR analysis of SKP2 in alveolar tissues of mice treated with CS or saline. (D) Detection of SKP2 and Beclin1 protein after MH‐S cells were transfected with siCtrl or siSKP2. (E) Detection of SKP2 and Beclin1 protein after MH‐S cells were transfected with siCtrl or siSKP2 and treated with protein synthesis inhibitor CHX. (F) Detection of Beclin1 and myc‐SKP2 proteins after overexpressing myc‐SKP2 in the MH‐S cell line for Co‐IP using Beclin1 antibody. (G) Detection of Beclin1, myc‐SKP2 and HA‐Ub using Beclin1 antibody after MH‐S cells were transfected with vectors encoding myc‐SKP2 and either wild‐type HA‐tagged ubiquitin or a lysine‐less mutant of HA‐tagged ubiquitin (Ub). *p < 0.05 vs. MH‐S cells treated with siCtrl (panel E); **p < 0.01 vs. normal mice (UT) (panel A) or mice treated with saline (panel B); and ***p < 0.001 vs. mice treated with saline (panel C). Measurement data were analysed by unpaired t test between two groups. Comparisons between multiple groups with different time points were performed using two‐way ANOVA. Bonferroni test was used for post hoc analysis. The experiment was conducted 3 times independently