Fig 2: Evaluation of deleting theC-terminuson STAM1 ubiquitination. Ubiquitination reactions as described in Experimental procedures were performed with a truncated variant of STAM1 (ΔC-tail) that is still able to bind to β-arr1. Reactions were incubated with 40 nM WT β-arr1 (WT) or preactivated variant of β-arr1 (R169E) or vehicle (n/a) in the presence or absence of 1 mM ATP with WT ubiquitin (WT Ub) (A) or lysine-less ubiquitin (0K Ub) (D). Reactions were terminated in 2× sample buffer immediately (0 min) or after incubation at 37 °C for 60 min. Reactions were analyzed by 7% SDS-PAGE and immunoblotting. Polyubiquitinated [Ub(n)], unmodified, monoubiquitinated (mono-Ub) STAM1 and nonspecific bands are indicated. Representative immunoblots are shown. STAM1 ubiquitination was quantified from the STAM (B and E) and ubiquitin (C and F) immunoblots using densitometry and shown as bar graphs. Data were expressed relative to the signal without βarr1 (“−“, minus symbol) and represent the mean ± S.D. from three independent experiments. Data were analyzed by one-way ANOVA GraphPad Prism. Adjusted p values are indicated. STAM, signal-transducing adaptor molecule.

Fig 3: Overexpression of ARRB1 suppressed LPS-induced ER stress in hepatic macrophages.A Double immunofluorescent staining for F4/80 and GRP78 in the indicated mouse liver sections. Nuclei were stained with DAPI in blue. F4/80 was stained green, and GRP78 was visualized red. The merged positive signals of F4/80 and GRP78 are visualized in yellow (scale bar: 50 μm). B, C GRP78 and CHOP mRNA levels in primary hepatic macrophages with LPS (0.5 μg/ml) challenge for 6 h from WT and ARRB1-KO mice were analyzed by real-time PCR. *P < 0.05 compared with the respective vehicle treatment; #P < 0.05 compared with LPS-induced WT mice by Student’s t test. D Protein expression levels of ARRB1, GRP78, eIF2α, p-eIF2α, CHOP, and ATF4 in ARRB1-overexpressing Raw264.7 and THP-1 cells with or without LPS (1 μg/ml) challenge for 6 h. E–H GRP78 and CHOP mRNA levels of ARRB1-overexpressing Raw264.7 and THP-1 cells with LPS (1 μg/ml) were challenged for 6 h and analyzed by real-time PCR. *P < 0.05 compared with the respective vehicle treatment; #P < 0.05 compared with vector cells following LPS treatment by Student’s t test. Data are presented as the mean ± SD of three separate experiments.

Fig 4: Summary of the proposed regulatory mechanism of ARRB1 in this study.ARRB1 suppresses LPS-induced release of inflammatory cytokines in response to activated macrophages. ARRB1 regulates the ER stress signaling pathway by inhibiting the phosphorylation of eIF2α and downstream ATF4 and CHOP expression in liver macrophages.

Fig 5: ARRB1 was involved in LPS-induced activation of liver macrophages.A Representative morphology images of Raw264.7 and THP-1 cells challenged with LPS at the indicated concentration for 6 h (scale bar: 50 μm). B Representative morphology images of Raw264.7 and THP-1 cells treated with LPS (1 μg/ml) were challenged at the indicated times (scale bar: 50 μm). C Western blotting analysis of ARRB1, TNF-α, IL-1β, and IL-6 in cells challenged with LPS at the indicated concentrations for 6 h. β-Actin was used as the loading control. D Western blotting analysis of ARRB1, TNF-α, IL-1β, and IL-6 in cells with LPS (1 μg/ml) challenge at the indicated times. E ARRB1 mRNA levels in Raw264.7 and THP-1 cells were analyzed at the indicated concentration for 6 h by real-time PCR. *P < 0.05 compared with LPS (0 μg/ml) treatment by one-way ANOVA. F ARRB1 mRNA levels in Raw264.7 and THP-1 cells with LPS (1 μg/ml) challenge at the indicated times were analyzed by real-time PCR. *P < 0.05 compared with vehicle treatment (0 h) by one-way ANOVA. G ARRB1 mRNA levels in mouse primary hepatic macrophages (PHMs) with LPS (1 μg/ml) challenge for 6 h were analyzed by real-time PCR. *P < 0.05 by Student’s t test. Data are presented as the mean ± SD of three separate experiments.