Figure 5

Regulation of Tollip expression by ethanol, HA35 and miR291b in primary cultures of Kupffer cells. (A) Kupffer cells isolated from ethanol- and pair-fed rats were cultured overnight and then treated with 100 µg/ml HA35 for 5 h and then challenged with 10 ng/ml LPS for an additional 60 min. (B,C) Kupffer cells isolated from ethanol- and pair-fed rats were nucleofected with either control hairpin or miR291b hairpin inhibitor 18 h post-nucleofection, Kupffer cells were treated or not with 100 µg/ml HA35 for 5 h and then challenged with 10 ng/ml LPS for an additional 1 hr. (A,B) Kupffer cells were lysed and Tollip expression measured by Western blot. HSC70 was used as a loading control. Images of the blots were cropped. Full size blots are shown in Supplemental Information Fig. 2. (C) Expression of TNFα mRNA was determined by qRT-PCR and normalized to 18 S. n = 4–5. Values represent means ± SEM. Values with different alphabetical superscripts are significantly different from each other, p < 0.05.