Fig. 3.

Influence of ATP8B1 depletion on IL-10/STAT3 signal transduction pathway in HMDM.

HMDM deficient for ATP8B1 were prepared as described in Fig. 2. The HMDM were stimulated with or without IL-10 (40 ng/ml) for 60 min and subjected to prepare whole cell lysates (a–d) or RNA (e). (a–c) Tyrosine and serine phosphorylation of STAT3. The prepared cell lysates (5 μg) were analyzed by immunoblotting (a). Expression level of each protein (b) and phosphorylation level of STAT3 (c, d) was quantified as described in the Supporting information. Data shown are means ± SEM of three independent experiments. α, STAT3α; β, STAT3β; pY,pS-α, pY,pS-STAT3α; pY-β, pY-STAT3β; pS-α, pS-STAT3α; BQL, below the limit of quantitation. (e) mRNA expression of STAT3-regulated genes. The isolated RNA was subjected to qPCR analysis. The expression of SOCS3, SBNO2, and ZNF36 in each reaction was normalized to that of ACTB. Data shown are means ± SEM of triplicate determinations. In (a, e), a representative result of three independent experiments is shown. *, P < 0.05, **, P < 0.01, ***, P < 0.001 versus siControl.