Western Blot Analysis of Tight Junction Proteins

Protein was isolated in situ, and western analysis performed as previously described [27 (link)]. Blots were probed using antibodies directed to Claudin-1, Occludin-1, ZO-1 (Thermo Fisher Scientific, North Ryde, Sydney, NSW, Australia), LC3, poly (ADP)-ribose polymerase (PARP), Sequestosome (Cell Signaling Technology, Boston, MA), Bcl2, NF-κβ (Santa Cruz Biotechnology, Dallas, TX) and β-actin (Sigma-Aldrich) followed by matching horseradish peroxidase-conjugated secondary antibodies (R&D Systems, MN, USA). Detection was performed using ECL Prime chemiluminescent substrate (GE Healthcare, Buckinghamshire, UK). Densitometry of histogram analyses was performed using Multi Gauge software (V3.1 Fugifilm, Tokyo, Japan). Density scores were normalized to both β-actin and the untreated control, and analyzed using a bootstrapped gamma regression model (log link) and stratified by replicate. The statistical analysis was performed using R statistical software (release 3.2.3) and results expressed as relative abundance.