Figure 4

Ubiquitylation of MFHAS1 by praja2 upregulates the expression of M1 macrophage polarization biomarkers and inhibits the expression of M2 macrophage polarization biomarkers. RAW264.7-MFHAS1-praja2 cells were transiently co-transfected with 2.75 μg plasmid expressing HA-MFHAS1 and 2.75 μg plasmid expressing Flag-praja2 plasmids, RAW264.7-MFHAS1 cells were transiently co-transfected with plasmid expressing 2.75 μg HA-MFHAS1 and 2.75 μg empty PCDH plasmid, and control cells were transfected with 5.5 μg empty PCDH plasmid. After 24 h, the cells were treated with or without Pam3CSK4 (10 ng/ml) for 6 h and were collected. Next, secretion of IL-6 and TNF-α was detected by performing ELISA (a and b). The mRNA levels of genes encoding M1 and M2 macrophage biomarkers IL-6, TNF-α, IL-1β, iNOS, Arg-1, MMR and IL-10 were quantified by performing qPCR and were normalized to the mRNA level of the gene encoding actin. All other groups were calibrated to 0 h control group (c–i). Values are expressed as means±S.D. of at least four independent experiments. *P<0.05, **P<0.01 or ***P<0.001