Casepase 3

Proteins were isolated from spinal cords of E21 fetuses by a lysis buffer (P0013B, Beyotime, China), and the protein concentration was quantified using the Bradford method. In total, 50 μg of protein extract was separated by 12.5% SDS-PAGE and then transferred with Tris-HCl methanol (20 mM Tris, 150 mM glycine, 20% methanol) onto polyvinylidene fluoride membranes (Millipore, USA) in a trans-blot electrophoresis transfer cell (Bio-Rad, USA). Blotting was probed with antibodies against cleaved CASEPASE3 (Cell Signaling Technology, USA), BCL2 (Sigma, USA), BAX (Cell Signaling Technology, USA), βШ-TUBULIN (Millipore, USA), SYNAPSIN11 (Millipore, USA), NF (Abcam, USA), or GAPDH (Proteintech, China). All immunoblots were run at least in triplicate. Immunopositive bands were visualized using enhanced chemiluminescence reagents (Millipore, USA). Detected bands were quantified by ImageJ software. The relative density of each protein was calculated by dividing the optical density value of each protein by that of the loading control (GAPDH).

Cells were collected and lysed on ice using a total protein extraction reagent (Beyotime Biotechnology, China). The protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Millipore, USA). The membrane was blocked in Tris-buffered saline with 5% (w/v) skimmed powdered milk, then incubated with primary antibodies overnight at 4°C, followed by an incubation with a horseradish peroxidase–conjugated secondary antibody for 1 hour at room temperature. Immunoreactive proteins were visualized using the Chemi Doc XRS Imaging System with an XRS camera (Bio-Rad, USA). Rabbit anti-mouse Bcl-2, Bax, Bcl-XL, and Casepase-3 antibodies were purchased from Cell Signaling Technology (New England BioLabs Inc. USA). Rabbit anti-mouse GPC-3 antibody was purchased from Affinity Biosciences (Changzhou, China).

Western blotting was performed as described previously 26 (link). Briefly, proteins were extracted by using RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 μg/ml leupeptin ,1 mM Na₂EDTA) added with phosphatase inhibitors and protease inhibitors (Biotool). The lysate was then centrifuged at 13000 rpm for 15 min at 4°C. The supernatants were collected and protein concentrations were measured using the BCA method. Antibodies against RIF1 were obtained from Santa Cruz Biotechnology and Bethyl Laboratories. Antibodies to BAX, BCL2, Casepase-3, PARP1, LIG4, Cyclin-D1, and c-Myc were purchased from Cell Signaling Technology. Antibodies against CDK2 and CDK4 were obtained from Santa Cruz. Antibody to β-actin was purchased from Sigma-Aldrich and used as a loading control.