A00166

Cellular protein extracts and/or spent medium were loaded onto a 12.5% polyacrylamide gel for SDS-PAGE analysis. After electrophoresis, the proteins were transferred to a nitrocellulose membrane using Trans-blot turbo (Bio-Rad, USA). The membrane was incubated in 5% (w/v) skimmed milk and 3% (w/v) BSA (NZYTech, Portugal) in phosphate buffered saline containing 0.1% (v/v) Tween 20 (PBS-T) for 1 h at room temperature (RT). The membrane was incubated with an antibody anti-cardosin B heavy-chain, henceforth referred as anti-cardosin B antibody (Antibody generated by HuCAL technology, Bio-Rad, USA) as previously described^{16 (link)} diluted in PBS-T (0.4 μg/ml) or anti-dsRed (sc-3909098, Santa-Cruz Biotech, USA) diluted 1:200 in PBS-T, for 1 h at RT and then at 4 °C overnight. Anti-human IgG peroxidase conjugated (A00166, GenScript, USA) or anti-mouse IgG peroxidase conjugated (A4416, Sigma Aldrich, USA) were used as secondary antibodies at dilutions 1:5000 and 1:4000 in PBS-T, respectively, under agitation for 2 h at RT. The signal was detected using Hyperfilm ECL (GE healthcare biosciences, USA) or Supersignal West femto maximum sensitivity substrate (Thermo Fisher Scientific, USA) in a ChemiDoc XRS+ system (Bio-Rad, USA). Protein molecular weight marker was NZYColour Marker II (NZYTech, Portugal) or Precision Plus Protein WesternC Standards (Bio-Rad, USA).