Anti rabbit antibodies

The 10630 (1:10,000) and GN60622 (1:10,000) neoepitope antibodies against the p20 subunit of active Casp616 (link) and Tub∆Casp623 (link), respectively, were generated in our laboratory. The β-actin clone AC-15 (1:5000, Sigma-Aldrich), Casp6 p10 clone B93-4 (1:250, BD Canada, Mississauga, ON, CA), Casp3, and full-length α-tubulin (1:1000, Cell Signaling Technology Inc., Danvers, MA, USA) antibodies were diluted in 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 (Sigma-Aldrich). Secondary anti-mouse (1:5000, GE Healthcare Life Sciences, Baie D’Urfe, QC, CA) and anti-rabbit antibodies (1:5000, Dako, Burlington, ON, CA) conjugated to horseradish peroxidase (HRP) were used to detect immunoreactive proteins using ECL prime western blotting detection reagent (GE Healthcare Life Sciences) and Kodak BioMax MR film (Kodak, Rochester, NY, USA). Secondary anti-mouse conjugated to alkaline phosphatase (Jackson Immunoresearch Laboratories Inc., West Grove, PA) was developed with nitro-blue tetrazolium (ThermoSci) and 5-bromo-4-chloro-3′-indolyphosphate (ThermoSci). The western blots were scanned with an HP scanner and densitometry was performed using ImageJ software (NIH, Bethesda, MD) by rendering tiff images into an 8-bit format and measuring the intensity values above background. Images were not modified except to adjust the contrast for the entire blot.

Total proteins from stably transfected MCF-7 and SUM159 cells were extracted with radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1% Triton, 0.1% SDS, 0.1% sodium deoxycholate and 1% Nonidet P40). 25 μg of total cellular proteins were resuspended in laemmli buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.004% bromphenol blue and 0.125 M Tris HCl, pH approx. 6.8) and heated at 95 °C for 5 minutes. Proteins were separated by a 4–15% Mini-PROTEAN^® TGX^™ Precast Gel (Biorad, Hercules, CA) and transferred onto a nitrocellulose membrane (Applichem, St. Louis, MO). The membrane was blocked for 1 hour with 3% non-fat dry milk in Tris buffered Saline/0.1% Tween-20. Immunoblotting was performed and antibodies specific for spinophilin (Cell Signaling, Danvers, MA, Cat.No. 9061S), the apoptosis marker PARP (Cell Signaling, Cat.No. 9542), pRb (directed against phosphorylated serine 807/8, Cell Signaling, diluted 1:1000 in 1% non-fat dry milk in Tris buffered Saline/0.1% Tween-20), and β-Actin (Sigma, Cat.No. A5441, clone AC-15) were detected using HRP-conjugated anti-mouse or anti-rabbit antibodies, respectively (Dako, Glostrup, Denmark). Visualization was performed using an enhanced chemoluminescence detection system (Super Signal West Pico, Thermo Scientific, Rockford, IL).

Immunohistochemical staining was performed as previously described [17 (link)]. Tissue sections of whole ear were deparaffinized and rehydrated with xylene and graded ethanol solutions. The sections were incubated with anti-IL-33 (Abcam, Cambridge, MA, USA) for 1 h in PBS and anti-NF-κB p65 (Cell Signaling Technology, Beverly, MA, USA) in PBS. After washing, the sections were incubated with anti-rabbit antibodies (Dako, Santa Clara, CA, USA) for 30 min and counterstained with Mayer’s hematoxylin. Images were taken using a light microscope at 200× (Nikon Instruments Inc.). Stained cells were counted using ImageJ.