Supersignal west pico detection kit

Total proteins from Candida infected epithelial cells were isolated as previously described with modifications [31 (link)]. TR146 epithelial cells were infected with each Candida strain at MOI (1:10). The Candida cells were allowed to infect epithelial cells for 3 h. After infection, the epithelial cells were lysed using 400μl RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing complete protease inhibitor cocktail (Roche, Basel, CH) and phosphatase inhibitors, left on ice for 30 min, then centrifuged for 10 min at 21000 × g at 4°C. Total protein concentration was estimated using bicinchoninic acid (BCA) assay (Thermo Fisher Scientific) and stored at −80°C. For immunoblotting, total protein (30μg) was separated by 12% SDS-PAGE and transferred to nitrocellulose membrane. Membranes were blocked in 5% milk or Bovine Serum albumin (BSA) in TBS containing 0.1% Tween-20 (TBST) at 23°C for 1 h. Blots were incubated in primary antibody (Phospo-MKP1 [S359] and c-Fos rabbit monoclonal antibodies (Cell Signaling Technology, Danvers, MA) for 16 h at 4°C. Membranes were washed twice with TBST and probed with a secondary antibody for 1 h at 23°C. Secondary antibodies were detected using SuperSignal West Pico detection kit (Thermo Fisher Scientific).

hOAS1 proteins expressed in bacteria or in mammalian cells were separated on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane at 100 V for 1 h. The membrane was incubated in blocking buffer (1 X Tris-buffered saline containing 5% non-fat dry milk and 0.1% Tween 20) at room temperature for 1 h and then cut into strips. To detect the hOAS1 isoform proteins expressed in bacteria, the membrane strips were incubated with anti-V5 (Sigma-Aldrich, St. Louis, MO, USA; 1:1000) antibody at 4 °C overnight. To detect overexpressed hOAS1 isoforms in HEK293 cell lysates, the membrane strips were incubated at 4 °C overnight with mouse anti-Flag antibody (Sigma-Aldrich, St. Louis, MO, USA; 1:1000) or mouse anti-β actin antibody (Cell signaling, Danvers, MA, USA; 1: 10,000). The membranes were washed three times for 10 min with 1 X Tris-buffered saline containing 0.1% Tween 20, and then incubated with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at room temperature. After washing, the membrane strips were processed for enhanced chemiluminescence using a Super-Signal West Pico detection kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol.

Immunoblotting was as described (12 (link)). Briefly, cells were seeded, treated as required and protein was harvested using RIPA buffer and a cocktail of protease/phosphates inhibitors (#A32963, Thermo Scientific). Protein was quantified using the BCA assay (#23227, Thermo Fisher Scientific) and 20-μg protein was resolved on appropriate SDS-polyacrylamide gels depending on the size of the protein of interest. Proteins were transferred onto Hybond-P (#GE10600023, Sigma) and probed using appropriate antibodies (see Supplementary Methods). Immunoreactivity was detected using the SuperSignal West Pico Detection Kit (#34577, Thermo Fisher Scientific). The level of protein signal was quantified using Image J.