Fig 2: CDG mediated both the MyD88‐ and TRIF‐dependent pathways in MPMs. (A) Schematic diagram showing the MD2‐TLR4 signalling pathway and downstream MyD88‐ and TRIF‐dependent branches. (B) MPMs from WT mice were stimulated with CDG (80 µM) for 5, 10, or 15 min. Representative immunoblots of co‐immunoprecipitation of MD2 and TLR4 in MPMs were shown. LPS (0.5 µg/mL, 5 min) was used as the positive control. (C) MPMs derived from WT mice were stimulated with CDG (80 µM) for 15 min. MD2 blockade was achieved using MD2‐neutralizing antibody (anti‐MD2, 100 ng/mL). Representative co‐immunoprecipitation images of TRIF/TLR4/MD2 and MyD88/TLR4/MD2 were shown. (D–H) MPMs from WT or MD2KO mice were treated with CDG (80 µM) for 1 h. Activation of MyD88‐ and TRIF‐dependent pathways was measured. Unphosphorylated proteins and/or GAPDH were used as the loading controls (n = 3 in each group, biological replicates). Representative blots (D, E) and densitometric quantification (F–H) are shown. (I, J) MPMs from WT mice were stimulated with CDG (80 µM) for 24 h. The protein levels of ICAM1 and VCAM1 were examined via Western blot analysis. GAPDH was used as the loading control (n = 3 in each group, biological replicates). Representative blots (I) and densitometric quantification are shown (J). Data information: Data are presented as mean ± SEM. One‐way ANOVA followed by Dunnett's multiple comparisons test.