Antibodies for cleaved caspase 3

Total protein was extracted from samples with RIPA lysis buffer, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto 0.45 μm PVDF membranes (Millipore). The following primary antibodies were used in the assays: antibodies for cleaved caspase-3 (no. 9654; Cell Signaling Technology, Danvers, MA, USA), GRP78 (no. 3183; Cell Signaling Technology), CHOP (no. 2895; Cell Signaling Technology), LC3A/B II/I (no. 12741; Cell Signaling Technology), p62 (no. 5114; Cell Signaling Technology), ATG-5 (no. 12994; Cell Signaling Technology), ATG-7 (no. 8558; Cell Signaling Technology), mTOR (no. 2983; Cell Signaling Technology), p-mTOR (Ser2448) (no. 5536; Cell Signaling Technology), p38 MAPK (no. 9212; Cell Signaling Technology), and p-p38 MAPK (no. 4511; Cell Signaling Technology), and β-actin (no. 3700; Cell Signaling Technology) was chosen as loading control.

Total protein was extracted from cell lines and mouse tumors using RIPA buffer (Thermo Fisher Scientific, MA, USA), and protein concentration was measured using bicinchoninic acid (BCA) assay. Total protein (50 μg) was separated by 13% SDS polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membranes were then blocked with 5% skim milk in Tris buffered saline with 0.1% Tween 20 (TTBS) and incubated with primary antibodies (1:1,000 dilution) including antibodies for cleaved-caspase3, cleaved-PARP, cyclinB1, cdc2, and β-actin (Cell Signaling, MA, USA) at 4°C overnight. After washing thrice with TTBS, the membranes were incubated with an HRP-conjugated secondary antibody (Cell Signaling Technology). Finally, the membranes were treated with ECL substrate (Thermo Fisher Scientific, MA, USA), and detection was conducted using a chemiluminescence detector (fusion solo S; Vilber). β-actin was used as loading control.

RVs were homogenized and protein gel electrophoresis samples were prepared as previously described [21 (link),25 (link)]. Equal protein amounts of samples were electrophoresed through a reducing SDS polyacrylamide gel and electroblotted onto a nitrocellulose membrane. Membranes were blocked and incubated with antibodies for cleaved caspase-3 (Cell Signaling Technology, Inc., Danvers, MA, USA), glutaraldehyde-3-phosphate-dehydrogenase (G3PDH), and collagen A1 (COLA1) (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and levels of proteins were detected by using horseradish peroxidase-linked secondary antibodies and an Enhanced Chemiluminescence System (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA).

The immunohistochemistry (IHC) was performed as previously described by us28 (link)29 (link). Briefly, fixed tumor tissues were dehydrated and embedded in paraffin. Tissues were sectioned into 5 μm thick sections using microtome (Leica Microsystems Inc., Buffalo Grove, IL). The sections were deparaffinized as well as rehydrated using xylene, ethanol and double-distilled water washes. Unmasking of antigen was done by boiling the sections in 10 mM sodium citrate buffer (pH 6.0). Sections were washed and incubated in 3% hydrogen peroxide solution. The sections were blocked with 5% goat serum and incubated with primary Antibodies for cleaved caspase 3 (1:300), LC3B (1:1000) and p62 (1:100) overnight at 4 °C. Next day the slides were stained using Ultravision ONE HRP polymer kit as per the manufacturer’s instructions (Thermofisher scientific, Fremont, CA). Counterstaining of sections was performed with Mayer’s hematoxylin and dehydrated. The slides were mounted using Permount (Fisher scientific, Fair Lawn, NJ) and imaged using Olympus microscope (Olympus America Inc, Center Valley, PA). Antibodies for cleaved caspase 3 (Cat.#9661S), LC3B (Cat.#3868S) and p62 (Cat.#5114S) were purchased from Cell Signaling. TUNEL assay was carried out according to manufacturer’s protocol (TUNEL assay kit was purchased from Calbiochem, San Diego, CA, USA).