Rabbit anti caspase 7

BCA (purity, 95.8%) was purchased from the Institute for Korea Traditional Medical Industry (Daegu, Korea) and dissolved in DMSO (Sigma-Aldrich, St. Louis, MO, USA). A BCA stock solution of 40 mM was stored at -80°C. Mouse anti-β-actin (1 : 5000 dilution), rabbit anti-p-AKT (1 : 1000 dilution), rabbit anti-AKT (1 : 1000 dilution), rabbit anti-p-p53 (Ser15) (1 : 1000 dilution), rabbit anti-p-p53 (Ser20) (1 : 1000 dilution), rabbit anti-p-p53 (Ser46) (1 : 1000 dilution), and rabbit anti-MDM2 (1 : 1000 dilution) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-p21 (1 : 1000 dilution), rabbit anti-p27 (1 : 1000 dilution), rabbit anti-p53 (1 : 1000 dilution), rabbit anti-FOXO3 (1 : 1000 dilution), rabbit anti-Bcl-2 (1 : 1000 dilution), rabbit anti-Bcl-xL (1 : 1000 dilution), rabbit anti-Bax (1 : 1000 dilution), rabbit anti-cleaved caspase-3 (1 : 1000 dilution), rabbit anti-caspase-3 (1 : 1000 dilution), rabbit anti-cleaved caspase-7 (1 : 1000 dilution), rabbit anti-caspase-7 (1 : 1000 dilution), rabbit anti-cleaved caspase-9 (1 : 1000 dilution), rabbit anti-caspase-9 (1 : 1000 dilution), rabbit anti-cleaved PARP (1 : 1000 dilution), and rabbit anti-PARP (1 : 1000 dilution) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Cells were lysed with RIPA buffer, and protein concentration was determined by BCA assay (Pierce, Waltham, MA). Protein lysates were separated by SDS–PAGE, transferred to nitrocellulose membranes (Bio-Rad), blocked with 5% milk/TBS, and probed with antibodies in 4°C overnight. The following antibodies were used for Western blot: mouse anti-CRT (Enzo Life Sciences, Farmingdale, NY), mouse anti-GAPDH (Fitzgerald, Acton, MA), rabbit anti-caspase 3 (Cell Signaling Technology, Danvers, MA), rabbit anti-caspase 7 (Cell Signaling Technology, Danvers, MA), rabbit anti-BAX antibody (abcam, Cambridge, MA), rabbit anti-PARP (Cell Signaling Technology, Danvers, MA), mouse anti-PARP C2-10 (Trevigen, Gaithersburg, MD), mouse anti-p53 (Santa Cruz, Dallas, TX), rabbit anti-CaMKII and anti-phospho CaMKII (Cell Signaling Technology, Danvers, MA).

Primary mouse antibodies used for western blotting and immunostaining were rat anti-K8 and rat anti-K19 (Troma I respectively Troma III, Hybridoma bank, Iowa, USA), mouse anti-K7 (RCK 105, Abcam, Cambridge, UK), mouse anti-K20 (IT-Ks 20.10, Progen, Frankfurt, De), rat anti-K18 (Troma II, Hybridoma bank, Iowa, USA) [24 (link)], mouse anti-tubulin (Sigma, Munich, Germany), rabbit anti-caspase-7 and anti-cleaved caspase-7 (Cell Signaling, Danvers, MA, USA), rabbit anti-MPO (Thermo Scientific, Waltham, MA, USA) and rat anti-Hsc70 (Stressgen, Victoria, Canada). The secondary antibodies used for staining were Alexa 488 or Alexa 546 anti-mouse, Alexa 488 anti-rat and Alexa 488 anti-rabbit antibodies (Invitrogen, Carlsbad, CA, USA). The secondary antibodies used for western blotting were: anti-mouse HRP (GE Healthcare, Little Chalfont, UK), anti-rabbit HRP (Cell Signaling, Danvers, MA, USA) and anti-rat HRP (GE Healthcare, Little Chalfont, UK) antibodies. Nuclei were stained with Draq5 (Cell Signaling, Danvers, MA, USA) or Dapi (Invitrogen Carlsbad, CA, USA). Antibodies used for FACS analysis were anti-CD4-FITC and anti-CD49d-PE or anti-L-selectin-PE (Immunotools, Friesoythe, Germany).

Protein preparation, SDS-PAGE and western blot analysis were conducted as described [35 (link)]. Rabbit anti-GAPDH (AbFrontier, Acris Antibodies, Herford, Germany), rabbit anti-heme oxygenase-1 (HO-1; Enzo Life Sciences, Lörrach, Germany), rabbit anti-caspase-7 (Cell Signaling Technology, New England Biolabs GmbH) antibody (in 20 mM Tris, 138 mM NaCl, pH 7.6, 5% (w/v) BSA, 0.1% (v/v) Tween^® 20), or rabbit anti-caspase-1 (Santa Cruz Biotechnology, Heidelberg, Germany, in 1x Roti^®-Block), and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (in 1x Roti^®-Block, Cell Signaling Technology) were used as primary and secondary antibodies, respectively.

Antibodies used for immunoblotting in this study were: rabbit anti-GSDMD antibody EPR19829 (Abcam, Cambridge, UK), rabbit anti-DFNA5/GSDME antibody EPR19859 (Abcam,), rabbit anti-γHexokinase #4959–9988 (Bio-Rad, Hercules, CA, USA), mouse anti-FLAG T0003 (Affinity Biosciences, Cincinnati, OH, USA), rabbit anti-caspase-1 (A-19) (Santa Cruz Biotechnology, Dallas, TX, USA), rat anti-caspase-2 #MAB3507 (Merck, Darmstadt, Germany), mouse anti-caspase-3 (clone 19) (BD BioSciences, Franklin Lakes, NJ, USA), rabbit anti-caspase-4 #4450S (Cell Signaling Technology, Danvers, MA, USA), mouse anti-caspase-5 (4F7) MBL International, Woburn, MA, USA), rabbit anti-caspase-6 (ARC0031 (Thermo Fisher Scientific, Waltham, MA USA), rabbit anti-caspase-7 (#9492) (Cell Signaling Technology), mouse anti-caspase-8 (1C12) (Cell Signaling Technology), rabbit anti-caspase-9 (#9502) (Cell Signaling Technology), rabbit anti-mouse IgG-HRP #A9044 (Sigma Aldrich, Burlington, MA, United States), donkey anti-rabbit-HRP #NA934 (Amersham, Little Chalfont, UK).
Q-VD-OPh was purchased from SMBiochemicals (Anaheim, CA, USA). Disulfiram was purchased from Selleckchem (Houston, TX, USA). TC13172 was purchased from MedChemExpress (Monmouth Junction, NJ, USA). All drugs were dissolved in dimethyl sulfoxide (DMSO) at 10 mM. The final concentration of the DMSO solvent in the assays was 1% regardless of drug concentration.