Figure 7.

CLT induces ubiquitin-mediated ECM1 degradation. (A) The mRNA level of ECM1 in LPS-induced Raw 264.7 cells treated with CLT. (B) 3D Docking conformations of ECM1 and CLT. (C) Raw 264.7 cells under LPS exposure were incubated with CLT or DMSO, and cellular thermal shift assays (CETSA) analyzed the thermal stabilization of ECM1 protein at different temperatures. The line graph presentation of quantitative analysis of ECM1 protein expression. (D) Raw 264.7 cells were treated with CLT under LPS exposure, treated with CHX (200 ng/mL), and collected at the indicated times for western blotting. The line graph presentation of quantitative analysis of ECM1 protein expression. (E) Representative western blots and quantitative analysis of ECM1. Raw 264.7 cells under LPS exposure were pretreated with MG132, followed by treatment with CLT. (F) Raw 264.7 cells under LPS exposure were treated with CLT and MG132. ECM1 was immunoprecipitated with an anti-ECM1 antibody, and the immunoprecipitates were probed with an anti-ubiquitin (UB) antibody. Statistical differences between two groups were analyzed by student's t-test analysis, and differences in multiple groups were determined by one-way ANOVA followed by Tukey's multiple comparisons. All data were presented as mean ± SD, n=3 for each group. *: p<0.05, **: p < 0.01, vs. CON OR LPS. ##: p < 0.01, vs. CLT OR LPS-CLT.