Figure 7: TJ-M2010-2 down-regulates the TLR/MyD88 signaling pathway.
(a) HK-2 cells were co-transfected with HA-MyD88/Flag-MyD88 or HA-MyD88/Flag-con. Co-IP assays were performed the same as with HEK293T cells (one of three independent experiments). (b) Densitometric analysis of Co-IP assays. The density of each HA-MyD88 lane was divided by that of Flag-MyD88 (*p < 0.0001 versus Control; ※p < 0.01 versus 0 μM; #p < 0.0001 versus 0 μM). Results are expressed as mean ± s.d. (c) Mice were exposed to IRI for 80 min with uninephrectomy (three mice were sacrificed for each group). Total proteins were extracted from kidney one day after IRI. TLR4, MyD88, p-IRAK4, TRAF6, p-p38, p-JNK and p-ERK protein levels were analyzed by Western Blot (one of three independent experiments). (d) Total proteins were extracted from HK-2 cells exposed to H/R injury. TLR4, MyD88, p-IRAK4, TRAF6, p-p38, p-JNK and p-ERK protein levels were analyzed by Western Blot (one of three independent experiments). (e) Nuclear proteins were extracted from HK-2 cells exposed to H/R injury. EMSA assay was used to detect NF-κB activity (one of three independent experiments). (f) Densitometric analysis of the NF-κB band in EMSA. (*p < 0.0001 versus Control; ※p < 0.01 versus H/R; #p < 0.0001 versus H/R). Results are expressed as mean ± s.d. (g) Three mice were sacrificed for each group. Renal tissues from short-term observation (80 min with uninephrectomy, day 1) and long-term observation (60 min without nephrectomy, day 28) were stained with MyD88 (red) and nucleus (blue).
