Caspase 3

The voucher specimens (Pisum sativum L. var. saccharatum Poir: CMU-104-PS-003) were deposited in Herbarium of College of Pharmacy, China Medical University, Taichung, Taiwan. Antipain, aprotinin, dithiothreitol, ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, leupeptin, Nonidet P-40, pepstatin, phenylmethylsulfonyl fluoride, sodium deoxycholate, trigonelline, and 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) were purchased from Sigma Chemical Company (St. Louis, MO). Antibodies to various proteins were obtained from the following sources: β-actin antibody was purchased from Sigma Chemical Company; Caspase-9, catalase, p38 (pThr180/Tyr182), and Raf (pSer259) were purchased from Abcam (Cambridge, MA); Mn-SOD and Cu/Zn-SOD were from Calbiochem (San Diego, CA); ERK (pThr202/Tyr204) was from ThermoFisher Scientific, Inc. (Waltham, MA); Nrf2 (pSer40) was from GeneTex, Inc. (Irvine, CA); Caspase-3 and protein kinase Cα (PKCα) from BD Biosciences (San Jose, CA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse and -rabbit IgG were from Abcam.

Mice were perfused with phosphate‐buffered saline and 4% paraformaldehyde. Brain and spinal cord were paraffin‐embedded and transverse sections were stained with Luxol Fast Blue and periodic acid Schiff (LFB/PAS) or hematoxylin and eosin (H&E). CNS tissue was further evaluated after labeling with antibodies against APP (1:2000 dilution, Merck Millipore), B220 (1:200, BD Bioscience), caspase‐3 (1:150, BD Bioscience), CD3 (1:200, Bio‐Rad Laboratories Inc., CA, USA), Mac‐3 (1:200, BD Bioscience), Nogo‐A (1:50, Santa Cruz) and Olig2 (1:300, IBL).

Frozen tissues were powdered under liquid nitrogen with the Multi-beads shocker and resuspended in 6 mol/L urea, 2 mol/L thiourea, 3% CHAPS, and 1% Triton X-100. The supernatant was then cleared by centrifugation at 15,000 rpm for 30 min. Cultured cells were washed with PBS, fixed with 10% TCA for 30 min, and lysed in the same buffer for 30 min. The supernatant was collected after centrifugation for 30 min.
Proteins were separated on SDS-polyacrylamide gels and transferred to a nitrocellulose membrane (Bio-Rad). After blocking for 1 h in Tris-buffered saline and Tween 20 (TBS-T) supplemented with 5% nonfat milk, membranes were incubated overnight at 4°C with primary antibodies against Bak1 (monoclonal, 1 : 250, MBL), PARP (1 : 1000, BD), Caspase-3 (1 : 1000, BD), Caspase-9 (1 : 1000, MBL), Caspase-2 (1 : 1000, BD), BMF (polyclonal, 1 : 1000, MBL), PUMA (monoclonal, 1 : 1000, BD), Bcl-2 (monoclonal, 1 : 500, BD), PP2A-catalytic α (1 : 1000, BD), MCL-1 (1 : 1000, BD), and β-actin (1 : 1000, BD). Membranes were then extensively washed with TBS-T and labeled with horseradish peroxidase-conjugated secondary anti-mouse (1 : 1000, GE) or anti-rabbit IgG (1 : 2000, GE). After additional washes with TBS-T, antigen-antibody complexes were visualized with ECL-Prime Kit (GE).

Western blotting was performed as previously described (28 (link)). The following primary antibodies were used: Caspase-3 (1:2,000, BD Biosciences) and beta-actin (1:5,000, Sigma-Aldrich). Densitometric analysis was performed on scanned blot images. Images were transformed to gray-scale on ImageJ software (v.1.50i, National Institutes of Health, USA). For each blot, a lane normalization factor was calculated by dividing the signal of each loading control band with that of the highest signal of loading control on the blot. The calculated lane normalization factor was then used to normalize caspase 3 band signals.

Total protein was isolated from the ipsilateral hemisphere samples, and the amount of protein was measured using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, USA). The procedure of Western blotting was performed as previously described [62 (link)]. The primary antibodies were as follows: CD63 (1:1000), Tsg101 (1:1000), CD206 (1:1000) (Abcam, Shanghai, China); caspase-3 (1:1000), cleaved caspase-3 (1:1000), ARG (1:200), Notch1 (1:500), and GAPDH (1:1000) (BD Biosciences, San Jose, USA). Proteins were determined using enhanced chemiluminescence (MilliporeSigma, Burlington, USA) and photographed using a Molecular Imager VersaDoc 4000 system (Bio-Rad, Hercules, USA).

Whole brain lysate from wildtype and knock-out animals was analyzed by Western blot for validation of gene knock-out using the following antibodies: Bax N20 (Santa Cruz, SC-493, 1:1000); Caspase-3 (BD Biosciences, 559565, 1:1000); Sarm1 (Abcam, ab226930, 1:1000); beta-Actin (Sigma, A5316, 1:1000).