Fluorchemm

Following the aforementioned treatments, RNA was isolated using Trizol (Sigma, St. Louis, MO, USA) according to the manufacturer’s guidelines. Once RNA was isolated, cDNA was synthesized using a ThermoFischer Scientific cDNA synthesis kit (ThermoFisher, Waltham, MA, USA). RT-PCR was performed using BAX and BCL-2 oligo primers as well as GAPDH for a control (Table 1). The PCR products were separated on a 1.5% agarose gel, using ethidium bromide as the indicating agent. Gels were imaged using a FluorChemM (Cell Biosciences, Palo Alto, CA, USA). Band intensities were calculated using FluorChem software. BAX and BCL-2 intensities were normalized to GAPDH.

Skimmed milk (10 µL) or MEC lysates (20 µg of protein) were prepared in Laemmli sample buffer containing 100 mM dithiothreitol (DTT), electrophoresed and immunoblotted as previously described (3,10). The following antibodies were used: anti-4 hydroxynonenal (4-HNE, 1:1000; Abcam), anti-mucin-4 (1:200; Santa Cruz Biotechnology), anti-endoplasmin (1:1000; Abcam), anti-phospho-serine (1:1000; Sigma-Aldrich), anti-HA (1:1000; Roche Applied Scientific), anti-phospho-STAT3 (1:1000; Cell Signaling), anti-STAT3 (1:1000; Cell Signaling), anti-E-cadherin (1:100; Sigma) and anti-ZO-1 (1 µg/mL; Life Technologies). Antibodies were detected with horseradish peroxidase-conjugated anti- rabbit or anti-mouse IgG (GE Healthcare) or anti-goat IgG (Pierce). Membranes were stripped before re-probing with another antibody or β-actin (1:5000, Sigma-Aldrich) as loading or normalization controls where indicated. Protein was detected with SuperSignal Femto Chemiluminescent Detection System (Pierce) and imaged using digital imaging (FluorChem M, Cell Biosciences, USA). Band signal intensity was quantified using AlphaView software (ProteinSimple, San Jose, CA).

Protein lysates were collected using M-PER mammalian protein extraction reagent with protease and phosphatase inhibitors added (Thermo Scientific; Rockford, IL). Lysates were cleared at 10,000 rpm for 10 min at 4°C then transferred to new tubes and frozen at -20°C until analysis. Protein concentration was determined using a BCA assay. Samples were combined with sample running buffer and boiled for 10 min. Proteins were separated on a 4–20% gel, transferred to nitrocellulose then probed using anti-β-actin and anti-Notch2 followed by HRP-conjugated secondary antibodies (all Cell Signaling Tech). Target proteins were detected using the chemiluminescent reagent Lumiglo (Cell Signaling) and visualized using FluorChemM (Cell Biosciences; Wallingford, CT).