Fig 2: The effect of IL-6 on NF-κB and Sp1/Sp3 levels and interactions in rat primary hepatocytes. (A–C) EMSA was performed to detect the binding levels of NF-κB and Sp1/Sp3 to Cbs in nucleus of rat primary hepatocytes with the treatment of 1 ng/ml IL-6 for 1 h and 150 µM quercetin for 3 h. Negative control: no protein extract for DNA to bind, thus there was only an unshifted probe band. (D) Western blotting detected tyrosine phosphorylation protein level of Sp3 and NF-κB in the nucleus of rat hepatocytes under IL-6 and quercetin treatment. (E–G) Co-immunoprecipitation (Co-IP) was performed to demonstrate directly that IL-6 promotes tyrosine phosphorylation of Sp3 and NF-κB in the nucleus of rat primary hepatocytes. (H–K) Effect of IL-6 and quercetin on NF-κB and Sp1/Sp3 level in nucleus of rat primary hepatocytes via western blotting. (L) The interaction of NF-κB and Sp3 in nucleus of rat primary hepatocytes was showed by Co-immunoprecipitation (Co-IP). n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 3: The activation of Sp3 mediated by NF-κB resulted in the inhibition of Cbs transcription in the nucleus of rat hepatocytes. (A–D) Western blotting showed that treatment with 30 µΜ JSH-23 for 1 h reversed the nuclear levels of NF-κB and Sp3 in rat primary hepatocytes with IL-6 stimulation. (E) Effect of 30 µΜ JSH-23 on Cbs mRNA levels in rat primary hepatocytes. (F, G) ChIP showed the regulation of Sp3 on Cbs transcription in IL-6-treated hepatocytes. n = 3. *p < 0.05, **p < 0.01.