Tubacin pretreatment suppresses 2ME-induced phosphorylation of HSP27 and MLC and alleviates 2ME-induced barrier dysfunction. A) HPAEC monolayers pretreated with vehicle control or 5μM tubacin for 1h, and then challenged with vehicle control (0 min) or 20 μM 2ME for the time indicated were extracted and analyzed by Western blot with anti-acetyl-tubulin, anti-beta-tubulin, anti-phospho-HSP27, anti-HSP27, and anti-diphospho-MLC antibodies. B) HPAEC monolayers grown on gold microelectrodes were pretreated with tubacin or vehicle control for 1h, then challenged with 20 μM 2ME for 15min (black columns) or 60 min (grey columns). TER decrease was presented as fold of control (2ME-induced TER decrease in the absence of tubacin). Shown are mean±SEM (n=3). C) Mice subjected to intravenous administration of vehicle control, 15 mg/kg 2ME, 0.3 mg/kg tubacin, and simultaneous administration of 2ME and tubacin for 3h were analyzed for EBD-albumin accumulation in the lung tissue. Shown are mean±SEM, number of animals is indicated on the figure. *p<0.05 are considered significantly different.