Total caspase 8

Paraffin (3 μm) sections of pancreas samples were stained with Hematoxylin/Eosin, Masson trichrome, or various primary and secondary antibodies. Paraformaldehyde (4%) fixed and paraffin embedded tissues were stained on an Autostainer Link 48 (Dako, Glostrup, Denmark). Primary antibodies used in this study were: Human/Mouse Gkn1 antibody from bio-techne R&D Systems (AF7287), anti-Gastrokine 2 from Abcam (ab188866), GAPDH from Santa Cruz (sc-25778), α-Tubulin from Cell Signaling (11H10), α-SMA from Cell Signaling (2125), Cleaved Caspase-3 from Cell Signaling (9661 S), Cleaved Caspase-8 from Cell Signaling (8592), total Caspase-8 from Cell Signaling (4927), FAS from Santa Cruz (sc-1024), Ki-67 from Abcam (16667), Bcl-xl from Cell Signaling (2762), Bcl-2 from Cell Signaling (2876), Mcl-1 from Cell Signaling (5453) and γH2AX from Novus Biologicals (NB100-384). Secondary antibodies used in this study were: anti-Rabbit from Dako EnVision+ System-HRP (K4011), rabbit-anti-Sheep Immunoglobulins/HRP from Dako (P0163).

The heart homogenates protein was separated on 10 or 12% SDS-polyacrylamide gels and transferred into Immobilon-P membranes (Merck, Darmstadt, Germany). Membranes were incubated in 5% (w/v) dried skimmed milk powder in Tris—buffered saline (pH 7.4) containing 0.1% Triton X—100 (TBST) buffer for 1 h at room temperature. After that, membranes were probed overnight at 4 °C with the appropriate primary antibodies for phosphorylated p38 (Cell signalling Technology, Danvers, MA, USA), #9211), total-p38 (Cell signalling Technology, Danvers, MA, USA, #8690), Bax (Santa Cruz Biotechnology, Inc., Dallas, TX, USA, #sc—493), cleaved-caspase 3 (Cell signalling Technology, Danvers, MA, USA, #9661), and total—caspase-8 (Cell signalling Technology, Danvers, MA, USA, #9746) (All of primary antibodies were diluted at 1:1000 in 1% (w/v) skimmed milk + TBST buffer) Then, the membranes were washed and exposed for 1 h at room temperature to horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit (Merck, Darmstadt, Germany, #AP132P) at 1:5000 in 1% (w/v) skimmed milk in TBST buffer. The signal was developed by exposure of the membranes with Affinity^® ECL Western Blot Kit, picogram Grade (Affinity Biosciences, Melbourne, Australia). The band intensity quantitation was captured and analysed by using ImageQuant™ LAS 500 (GE Healthcare, Chicago, IL, USA).

Total protein, cytosolic and nuclear protein were extracted from HK-2 cells using lysis buffer and NE-PER extraction kit (Thermo Scientific, Rockford, IL, USA) respectively, containing protease inhibitor cocktail (Sigma-Aldrich). For mouse kidney tissue, total protein were extracted by NucleoSpin Triprep or fractionized by the NE-PER extraction kit. Protein concentration was quantified by BCA Protein Assay Kit (Thermo Scientific). Equal amount of protein was resolved by 12% SDS-PAGE and then transferred to PVDF membranes. After blocking with 5% non-fat milk, the membranes were subjected to overnight primary antibody incubation using antibody against active β-catenin, total caspase-3, total caspase-8 (Cell Signalling Technology, Beverly, CA, USA), Dicckopf-3, TATA-binding protein (TBP; Abcam, Cambridge, UK) and β-actin (Thermo Scientific). The membranes were then incubated with peroxidase conjugated secondary antibodies (Dako, Carpinteria, CA, USA) accordingly and visualized with ECL prime chemiluminescnece (GE Healthcare, Buckinghamshire, UK) by using ChemiDoc XRS+ system (Bio-Rad, Hercules, CA, USA). Protein expression levels were quantified and normalized to β-actin/TBP by Image Lab software (Bio-Rad).