Chemiluminescent substrate

The total proteins from caudal fins of adult fish were extracted by incubation in RIPA lysis buffer (Sigma-Aldrich, Burlington, MA, USA) with protease cocktail (Sigma-Aldrich) and separated in sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred to polyvinylidene fluoride (PVDF) membrane (Merck Millipore Ltd, Tullagreen, Ireland) and blocked by 4% skim milk in Tris-buffered saline with 0.1% Tween-20 (TBST) v/v. Next, membrane was immunolabeled with anti-LRP5 rabbit monoclonal antibodies (1:1000, D80F2, Cell Signaling Technology, Danvers, MA, USA) or anti-α-Tubulin mouse monoclonal IgM antibodies (1:1200, TU-02, sc-8035, (Santa Cruz Biotechnology, Dallas, TX, USA). Secondary antibodies (1:1500, ab97240, Goat Anti-Mouse, and 1:1500, ab97051, Goat Anti-Rabbit, Abcam) were incubated for 1 hour at room temperature. Membranes were developed using chemiluminescent substrate (Advansta Inc., San Jose, CA, USA) and imaged via UVITEC Alliance Q9 Imager (Cleaver Scientific, Warwickshire, UK).

Saffron treated cells were lysed in RIPA buffer. Protein concentrations were quantified using the Pierce BCA Protein Assay (Thermo Fischer Scientific) according to manufacturer’s instructions. For MACC1 and DCLK1 30 μg, cleaved-caspase-3 and cleaved-PARP 60 μg protein lysates were separated using 12% sodium dodecyl sulfate polyacrylamide gels. The proteins were transferred to nitrocellulose membranes and incubated with the respective primary antibody followed by HRP-labelled secondary antibodies. Bound antibodies were detected by incubating the membranes in a chemiluminescent substrate (Advansta, San Jose, CA, USA). Protein bands were visualized by exposure to SuperRX X-ray films (Fujifilm, Tokyo, Japan). β-actin and vinculin were used as loading controls.