Figure 5.

Effects of RIPK2 PROTAC on LPS-induced pro-inflammatory gene expression, protein levels of iNOS and COX-2, and phosphorylated NF-κB p65 and MAPK p38 levels in microglia. SIM-A9 microglial cells were pretreated with 1 µM RIPK2 PROTAC for 4 h followed by 20 h incubation with 10 ng/mL lipopolysaccharide (LPS). After that, cells were harvested for RNA and protein extraction. (A) Graph shows that RIPK2 PROTAC partly reduced LPS-induced Ptgs2, Il-1β, Il6, Ccl2, and Mmp9 gene transcription but did not affect gene expression of Nos2 and Tnfα. (B–E) RIPK2 PROTAC had no effects on LPS-induced increase in iNOS protein levels (B,C), but it slightly attenuated LPS-induced upregulation of COX-2 levels (B,D). Treatment with RIPK2 PROTAC had no effects on increased levels of phosphorylated NF-κB p65 (B,E) or p38 MAPK (B,F) induced by LPS. At same time, LPS-triggered upregulation of RIPK2 was potently degraded by its PROTAC (B,G). qRT-PCR data are normalized to reference genes Cyc1 and Rltr2aiap and represented as fold changes compared to LPS treatment, and immunoblot data are normalized to β-actin. One-way ANOVA with Bonferroni post-test; * p < 0.05, ** p < 0.01, and *** p < 0.001. Data are represented as mean ± SEM from three independent experiments.