Membranes were probed with primary antibody (1:1,000) in 10 mL blocking buffer overnight at 4°C. After washing, membranes were further probed with appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (1:2,000) in 10 mL of blocking buffer for 1 hour at room temperature. SuperSignal Y-27632 price West Pico Chemiluminescent Substrates (Thermo Fisher Scientific, Rockford, IL) were used for chemoluminescence development. Results are shown as mean ± standard deviation (SD).

Statistical analyses were performed using unpaired Student’s t test with P < 0.05 (two-tailed) considered significant. We first determined whether IR stimulation triggers Gsk3β phosphorylation/dephosphorylation in mouse livers subjected to 90 minutes of warm ischemia, followed by

various lengths of reperfusion. Compared with sham controls, APO866 manufacturer the phosphorylated Gsk3β (at serine 9) level was slightly reduced by ischemia itself (time 0), and then rapidly increased at reperfusion and remained phosphorylated throughout the reperfusion phase (1-6 hours) (Fig. 1). As total Gsk3β levels were equal at all timepoints, these results indicate that the constitutively active Gsk3β in the liver was inactivated by the IR insult and sustained inactive thereafter. Gsk3β activation profile was distinct from that of MAP kinases, which were only transiently activated by IR, but quickly decreased to baseline by 4 hours postreperfusion (Fig. 1). The phosphorylation of tyrosine 216 on Gsk3β was not detected, nor was the phosphorylation of Gsk3α in IR livers (data not shown). Thus, liver most IR triggers Gsk3β phosphorylation on serine 9, which is sustained throughout the reperfusion period. To address the functional significance of the constitutively active Gsk3β, we treated mice with a Gsk3β-specific chemical inhibitor, SB216763, prior to the onset of ischemia. The inhibition of liver Gsk3 activity in vivo was

confirmed by the reduced phosphorylation level of glycogen synthase, the substrate of Gsk3β (Fig. 2A). As compared with vehicle-treated controls, livers in animals receiving SB216763 suffered less severe IRI, evidenced by significantly lower sALT levels (Fig. 2B, 7,709 ± 1,689 versus 2,946 ± 513; P = 0.0053), better preserved liver architecture by histology (Fig. 2C) and Suzuki grading (Fig. 2D, 3.6 ± 0.24 versus 2.2 ± 0.40 n = 5-6/group, P < 0.05). In parallel, pretreatment with SB216763 suppressed neutrophil activation, measured by MPO activity (Fig. 2E, 5.73 ± 1.50 versus 3.30 ± 0.54; P < 0.05), and diminished the induction of TNF-α, IL-1β, IL-6, and CXCL10 in IR-livers (Fig. 2F). It has been shown that IR-induced IL-12 and IL-10 are transient and occur early in postreperfusion.