C jun

Rat tissues or Bend.3 cells were homogenized by sonication in RIPA buffer. Protein concentrations were determined with a BCA assay kit (QPBCA, Thermo Scientific). Protein extracts were electrophoresed by 10% SDS-PAGE. The separated protein was then electrotransferred onto PVDF membranes, shaken slowly in blocking solution (5% milk in TBST) for 90 min and incubated overnight with antibodies against occludin (1:500, Invitrogen), claudin-5 (1:1000, Abcam), MAP3K2 (1:500, Invitrogen), p-JNK1/JNK2 (1:1000, Invitrogen), JNK (1:1000, Invitrogen), p-c-Jun (1:500, Invitrogen), c-Jun (1:500, Invitrogen), active caspase-3 (1:1000, Invitrogen), caspase-3 (1:2000, Invitrogen), and β-actin (1:5000, Millipore Sigma) as a loading control. Afterwards, the membranes were washed and transferred into a buffer with appropriate secondary antibodies for 1 h at room temperature. An ECL detection kit was used to visualize the protein bands. ImageJ (LOCI, Madison, WI, USA) software was used for quantitative analysis of western blot results.

Transient transfection of cells with 20 nM siRNA oligonucleotides or plasmids was performed using RNAiMAX or Lipofectamine 2000 (Invitrogen) according to the manufacturer's instruction with slight modifications. The siRNA IDs were as follows, ANGPTL4 (siRNA IDs: HSS181878, HSS181879); NOX4 (siRNA IDs: HSS121312, HSS121313); MMP-1 (siRNA IDs: HSS106609, HSS106610); MMP-3 (siRNA IDs: S8854, S8855); MMP-9 (siRNA IDs: S8862, S8863); c-Jun (siRNA IDs: HSS105641) (Invitrogen); Negative control siRNAs (siRNA IDs: D-001810-10-50) (Dharmacon, Lafayette, CO, USA). For use in transfection, 3.75 μl of RNAiMAX or Lipofectamine 2000 was incubated with siRNA or plasmid in 1.5 ml of Opti-MEM medium (Invitrogen) for 30 min at room temperature. Following the removal of Opti-MEM medium and replacement with 3 ml of fresh culture medium, cells were incubated for an additional 24 h, unless stated otherwise.