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. 2013 Jul 22;8(7):e68659. doi: 10.1371/journal.pone.0068659

Figure 2. Rk1 inhibits VEGF-induced destabilization of TJ proteins.

Figure 2

(A) HRECs were pretreated for 40 min with or without Rk1 (10 µg/ml) prior to stimulation with VEGF (20 ng/ml) for 6 h. Total mRNAs were isolated and RT-PCR was performed with specific primers for human ZO-1, ZO-2, and occludin. GAPDH served as an internal control. (B) Confluent HRECs were pretreated for 40 min with or without Rk1 (10 µg/ml) prior to stimulation with VEGF (20 ng/ml) for 1 h. Translocation of TJ proteins was assessed as described in “Experimental Procedures”. The Triton X-100-insoluble and soluble fractions were subjected to SDS-polyacrylamide gel electrophoresis followed by Western blot analysis with anti-ZO-1, anti-ZO-2, anti-occludin and anti-Actin. Blots are representative of three independent experiments. (C) Densitometric analyses are presented as the relative ratio of tight junction proteins in membrane and cytosol and quantified with NIH ImageJ software. Data are the mean ± S.E. **, p < 0.01 vs. Control. ##, p < 0.01 vs. VEGF alone.