Characterization of purified 26S proteasome after treatment with Torin1. (A, B) Effect of mTORC1 inhibitor Torin1 on proteasomes. HEK293 cells were treated with 250 nM Torin1 for indicated time periods. Whole-cell lysates (A) and purified proteasomes (B) were collected and separated on denaturing SDS-PAGE. (C) Comparison of phosphorylation of purified proteasomes using pan-phosphorylation antibodies (61-8300, Invitrogen, 1.0 μg/ml). (D, E) Effect of Torin1 treatment on proteasome activity. Purified proteasomes (2 ug) were separated on non-denaturing (native) PAGE, followed by in-gel suc-LLVY-AMC hydrolysis or immunoblotting against the 20S subunit, PSMA4, to visualize proteasomes (D). Protein samples (10 μg) were subjected to suc-LLVY-AMC (12.5 μM) hydrolysis to estimate proteasome activity (E).
Roles of heterogenous liver macrophages during the progression of liver injury. Under the pathologic process of liver injury, the resident embryonic-derived Kupffer cells (EmKCs) located inside the sinusoidal endothelium recognize microbial pathogen-associated molecular patterns (PAMPs) and damaged cell-released damage-associated molecular pattern (DAMPs) via PRRs and inflammasome. The activated EmKCs release inflammatory cytokines and CCL2 chemokine to recruit circulating monocytes. They develop into inflammatory Ly6Cpos monocyte-derived macrophages (MoMFs) or anti-inflammatory Ly6Cneg MoMFs to promote or suppress fibrotic activation of hepatic stellate cells (HSCs). MoMFs can fully differentiate toward monocyte-derived KCs (MoKCs) by activation of LXRα and ID3 transcription factors. TREM2 and CD9 positive lipid-associated macrophages (LAMs) are derived from MoKCs and their roles in the development of liver injury has not been identified yet. The stimulation, differentiation, and conversion of hepatic macrophages are indicated as shown colored arrows.
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