Association of Notch1 with ASGR1 in hepatocytes. (A) The relationship between Notch1 and ASGR1 in hepatocytes were evaluated by immunoprecipitation assay (representative images from 3 independent experiments). (B) Hepatocytes were isolated from wild-type mice, incubated with Asialofetuin (200 μg/mL) for 15 minutes, and then treated with desialylated platelets, followed by measuring Notch1 activation (cleaved Notch1 expression) (C), HES5 expression (D), STAT3 phosphorylation (E), as well as TPO production (F) by western blot (data were shown as mean ± SD; n = 3; 2-way ANOVA). (G) Wild-type mice were IV injected with clodronate liposomes 2 days before infusion of desialylated platelets to deplete macrophage Kupffer cells, and the depletion efficiency was evaluated by measuring the expression of macrophage marker F4/80 in the liver by western blot (representative image was shown from 5 independent experiments). (H) Twenty-four hours after the transfusion of desialylated platelets, liver was isolated to measure TPO mRNA expression by quantitative real-time PCR (mean ± SE; n = 5; 2-way ANOVA). The expression of cleaved Notch1, HES5, and TPO was quantified as a ratio relative to β-tubulin level, and the phosphorylation level of STAT3 was quantified as a ratio relative to total level. Data were shown as mean ± SD (n = 3; 2-way ANOVA). ∗∗P < .01; ∗∗∗P < .001. PCR, polymerase chain reaction.