Samples containing equivalent amounts of protein were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (7.5%-10%) and then were transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked for 2 h in a blocking buffer [Tris-buffered saline with 0.05% Tween 20 (TBST) containing 5% skim milk] and then incubated overnight at 4 °C with primary antibodies (claudin-5, 1:1000, Invitrogen, Carlsbad, CA, USA; occludin, 1:1000, Abcam, Cambridge, MA, USA; S1PR1, 1:1000, Abcam, Cambridge, MA, USA; p-extracellular regulated protein kinases (ERK) 1/2, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA; ERK 1/2, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA). After incubation with corresponding secondary antibodies at room temperature for 1 h, the protein bands were estimated by Immobilon Western Chemiluminescent horseradish peroxidase substrate. All protein bands were visualized by enhanced chemiluminutesescence (ECL) kit (EMD Millipore, Billerica, MA, USA) and were recorded by Tanon 5500 Chemiluminescence Imaging system (Tanon Science and Technology Co., Ltd., Shanghai, China). ImageJ software (US National Institutes of Health) was used to analyze the blots.

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