Ab32150

After treatment for 48 h, HepG2 cells were lysed with RIPA buffer containing complete Mini protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). After centrifugation, the supernatant was collected and stored at -80°C. Proteins were resolved by SDS–PAGE using commercially available 4–12% NuPAGE Bis-Tris gels (Invitrogen, Basel, Switzerland) and transferred using the Trans-Blot Turbo Blotting System (Bio-Rad, Cressier, Switzerland). The membranes were incubated with PARP (46D11) rabbit mAb (Cell Signaling Technology, Danvers, MA, United States), Anti-active Caspase-3 antibody (ab32042, Abcam, Cambridge, United Kingdom), Anti-pro Caspase 3 antibody (ab32150, Abcam, Cambridge, United Kingdom), Anti-SOD1 (ab20926, Abcam, Cambridge, United Kingdom) or Anti-SOD2 (ab16956, Abcam, Cambridge, United Kingdom) antibodies. After washing, membranes were exposed to secondary antibodies (Santa Cruz Biotechnology, Dallas, TX, United States). Immunoblots were developed using enhanced chemiluminescence (GE Healthcare, Little Chalfont, United Kingdom). Band intensities of the scanned images were quantified using the National Institutes of Health Image J program, version 1.48.

Total protein was extracted followed by concentration measurement utilizing a bicinchoninic acid kit. Protein (50 μg) was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane which was then blocked with 5% skimmed milk powder at ambient temperature for 1 h. Next, the membrane was probed with diluted primary rabbit antibodies to Smad2 (ab33875, 1: 1000, Abcam), Smad4 (ab40759, 1: 5000, Abcam), HDAC3 (ab7030, 1: 500, Abcam), TGIF1 (ab52955, 1: 5000, Abcam), cleaved caspase-3 (ab32042, 1: 1000, Abcam), total caspase-3 (ab32150, 1: 1000, Abcam), MMP-2 (ab97779, 1: 2000, Abcam), MMP-9 (ab38898, 1: 1000, Abcam), TGF-βRII (sc-17791, 1: 1000, Santa Cruz Biotechnology, Santa Cruz, CA) and GAPDH (ab9485, 1: 2500, Abcam) overnight at 4ºC. The next day, the membrane was re-probed with HRP-labeled secondary antibody of goat anti-rabbit antibody to IgG H&L (ab97051, 1: 2000, Abcam) for 1 h. The results were visualized utilizing enhanced chemiluminescence reagents (BB-3501, Ameshame, Little Chalfont, UK). Images were acquired through Bio-Rad image analysis system (Bio-Rad Laboratories, Hercules, CA), followed by analysis using Quantity One v4.6.2 software. The relative protein level was described as the ratio of gray value of protein to be tested to that of GAPDH [22 , 23 (link)].

Total protein was extracted from cell lines or human bone marrow samples using RIPA lysis buffer (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Next, the protein concentration was detected using the BCA protein assay (Beyotime Biotechnology, Shanghai, China). Proteins were separated by 10% SDS-PAGE and transferred onto PVDF membranes. The membranes were incubated with 5% non-fat milk at room temperature for 2 h, and then incubated with rabbit anti-mouse primary antibodies, including anti-Pro-caspase-3 (ab32150, Abcam), -cleaved-caspase-3 (ab2302, Abcam), -XIAP (ab28151, Abcam), -PIM-2 (4730T, Cell Signaling) and -β-actin (3700S, Cell Signaling) at 4°C overnight. Then the membranes were incubated with secondary antibodies (Beyotime, Shanghai, China) at room temperature for 1 h. β-actin was used as an internal standard. The protein bands were visualized by electrochemiluminescence (ECL) assay (Millipore, Billerica, MA, USA).

The University of Illinois Veterinary Diagnostic Laboratory database was reviewed for cases of canine astrocytoma, and identified tissues were recut for procaspase-3 immunohistochemistry. Processed slides were deparaffinized in xylene and rehydrated in alcohol. Endogenous peroxidase activity was blocked with Biocare #PX968 Peroxidazed 1 at room temperature for 5 minutes, rinsed with TBS wash buffer, and then incubated for 10 minutes at room temperature with Biocare #BP974 Background Punisher. Slides were incubated with procaspase-3 antibody (Abcam #ab32150) for 39 minutes at a dilution of 1:3000, washed, and then incubated with Rabbit-on-Canine HRP-Polymer (Biocare #RC542) for 30 minutes. Slides were washed in TBS wash buffer. The reaction was developed using DAB substrate for 5 minutes and the slides were then counterstained with Mayer's hematoxylin. Canine lymph nodes served as negative and positive controls.

Protein extracts (10 μg) prepared from LCLs or B lymphocytes were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Wisconsin, USA). The membranes were blocked with 5% milk in Tris-buffered saline (pH 7.6) containing 0.1% Tween 20 and incubated overnight at 4°C with specific primary antibodies against the following proteins: TNF receptor-associated factor 1 (TRAF1, 1 : 1000, ab203316, abcam, Cambridge, UK), IκB kinase (IKK)-α (1 : 10000, ab109749, abcam), NF-κβ-inducing kinase (NIK, 1 : 500, ab203568, abcam), P52 (1 : 2000, ab129097, abcam), pro-caspase 3 (1 : 1000, ab32150, abcam), caspase 3 (1 : 500, ab49822, abcam), poly (ADP-ribose) polymerase (PARP, 1 : 2000, ab74290, abcam), and β-actin (1 : 5000, 66009-1-Ig, Proteintech, USA). After three washes with PBS/Tween 20, the membranes were incubated with horseradish peroxidase-conjugated secondary goat antirabbit IgG (1 : 20000, PAB160011, Bioswamp) for 2 h at room temperature. Protein bands were visualized by enhanced chemiluminescence color detection (Tanon-5200, TANON, Shanghai, China) and analyzed using AlphaEase FC gel image analysis software.