Rabbit anti dsg2

Fenofibrate was purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit anti-Phospho-stat3 (Tyr705), rabbit anti-Phospho-SMAD3 (Ser423/425), rabbit anti-SMAD, rabbit anti-Phospho-AKT (Ser473), rabbit anti-AKT, rabbit anti-α-SMA, rabbit anti-Collagen I antibodies, mouse anti-stat3, and mouse monoclonal anti-β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). Rabbit anti-DSG2, rabbit anti-PPARα, rabbit anti-GAPDH antibodies were from Abcam Inc. (Cambridge, MA, USA).

Proximity ligation assays (PLA) were carried out as recommended by the manufacturer ((Sigma-Aldrich, Munich, Germany): In brief, cells were seeded on coverslips and treated with TNFα for 24h when reached confluency. Two primary antibodies from different species were selected. Following antibodies were used: mouse anti-Dsg2 (Invitrogen, Carlsbad, CA, USA) at a dilution of 1:100, rabbit anti-PI-3-kinase 1:100 (Cell signaling, Leiden, Netherlands), mouse anti-Plakoglobin 1:100 (Progen, Heidelberg, Germany) and rabbit anti-Dsg2 1:100 (abcam, Cambrige, UK). After blocking of unspecific binding sites, slides were incubated with the mentioned primary antibodies. Next, a pair of oligonucleotide-labeled secondary antibodies (PLUS and MINUS Probes) which bind to the primary antibody were applied. When the PLA probes are in close proximity, connector oligos join the PLA probes and become ligated by addition of ligase at a dilution of 1:40. As a consequence, a closed circle DNA template is formed and acts as a primer for a DNA polymerase. Finally, labeled oligos hybridize to the complementary sequences within the amplicon, which are then visualized as discrete spots (PLA signals) by microscopy analysis. As negative controls, the same procedure was carried without application of primary antibodies as recommended by the manufacturer.