Western blots of ZO-1 immunoprecipitated from hBMEC membrane fractions show phosphorylated serine/threonine (p-Ser/Thr) reactive blots and the corresponding ZO-1 reactive blots detected at the same position on the same PVDF membrane stripped and reprobed with ZO-1 antibody (top). Quantitative data showing the ratio of p-Ser/Thr to total ZO-1 compiled from multiple experiments are also shown (bottom). A: hBMECs treated with 100 ng/ml IL-1β for 30 min, 1.5 h or 6 h, show that total serine/threonine phosphorylation of immunoprecipitated ZO-1 is significantly increased at 1.5 h and 6 h (P < 0.05 and 0.01, respectively; n = 4). B: there is a significant decrease in p-Ser/Thr reactivity of ZO-1 following treatment with Gö6976 and IL-1β relative to IL-1β alone following 1.5 h (left; open bars) or 6 h (right; closed bars) of IL-1β treatment (P < 0.001; n = 4 or 10, respectively) (*P < 0.05, **P < 0.01, ***P < 0.001).