Htra2

An apoptosis inhibitor ZVAD (HY-16658) was obtained from MCE (Princeton, NJ, USA). Primary antibodies CTSH (sc-398527), FAS (sc-8009), Survivin (sc-17779), XIAP (sc-55550), DIABLO (sc-393118), and HTRA2 (sc-365594) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA); GAPDH (#5174S), AIF (#7495S), CASP9 (#9502), CASP3 (#9662), and TOM20 (42406S) were purchased from Cell Signaling Technology (Danvers, CO, USA); and anti-actin was purchased from Sigma Aldrich (Saint Louis, MO, USA, prod. no. A3853). Secondary antibodies goat antirabbit IgG- (H+L) HRP conjugate (cat. no. 170-6515) and goat anti-mouse IgG- (H+L) HRP conjugate (cat. no. 170-6516) were obtained from Bio-Rad Laboratories (Mississauga, ON, Canada).

For the immunoblotting, a small amount (10 μL) of the CPB solution (0.5 McF), was mixed with Laemmli Buffer at a ratio 4:1, and loaded on 1.0 mm NuPAGE® Novex® 4–12% Bis-Tris protein gradient gel. Normal human liver protein extract was used as a positive control. Proteins were separated by the SDS-PAGE at 100 V for 1 h and transferred at 30 V for 1.5 h 4 °C. Blots were probed with either fetuin-A (ab34505, Abcam) or albumin (ab9092, Abcam) antibodies. Horseradish peroxidase conjugated rabbit or goat secondary antibodies (Santa Cruz Biotechnology) were used at dilution 1:500. Proteins were visualised with enhanced chemiluminescence by using the Amersham ECL Prime detection reagent (General Electric Healthcare). For the experiments described in Figs 6e and 7a,b, total protein was extracted from cells using RIPA buffer (ThermoFisher) with protease inhibitor cocktail (Roche). Electrophoretically resolved protein samples were transferred and probed with cleaved caspase 9 (GTX86912, GenTex), xiap (sc-11426, Santa Cruz Biotechnology), htra2 (sc-15467, Santa Cruz Biotechnology), shh (sc-9024, Santa Cruz Biotechnology), ptch1 (sc-9016, Santa Cruz Biotechnology), and gapdh (sc-20357, Santa Cruz Biotechnology) antibodies.