Cleaved poly adp ribose polymerase cleaved parp

Three animals from each group were randomly selected, anesthetized and transcardially perfused with pre-chilled saline. A 4 cm segment of spinal cord including the injury site was quickly harvested and divided into three 1 cm segments on ice: 0.5cm rostral and caudal from the injury site, and the epicenter. Proteins were homogenized and extracted using RIPA buffer (Cell Signaling) with protease inhibitor cocktail (Roche). Protein lysates were electrophoresed on SDS polyacrylamide gel and transferred to PVDF membranes. After blocking, membranes were incubated with myelin basic protein (MBP, Millipore for cell and Cell Signaling for tissue), cleaved poly (ADP-ribose) polymerase (cleaved PARP), total Caspase 3, cleaved caspase 3, c-Myc, Actin and GAPDH (all from Cell Signaling) at 4 °C overnight. After rinsing, HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (both from Cell Signaling) were incubated for 2h. Chemiluminescent reagents were added, and signals were visualized using X-ray films.

Protein samples of cells were lysed in buffer containing 50 mM Tris–HCl, pH 7.6, 150 mM NaCl, 0.1% sodium dodecyl sulfate, 1% Nonidet P‐40, and 0.5% sodium deoxycholate, and western blotting was performed as previously described.⁹, ¹⁶ Quantification was achieved by densitometry using ImageJ software (National Institutes of Health).
Membranes were incubated with the following antibodies: Antibodies against ATP‐binding cassette sub‐family G member 2 (ABCG2; #4477), phosphorylated‐Cdc2 Tyr15 (p‐Cdc2; #9111), phosphorylated‐Chk1 Ser345 (p‐Chk1; #2348), cleaved poly ADP ribose polymerase (cleaved PARP; #5625), topoisomerase I (TOP1; #79971), and topoisomerase IIα (TOP2a; #12286) were purchased from Cell Signaling Technology. Antibodies against ATP‐binding cassette sub‐family B member 1 (ABCB1; #sc55510) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH; #sc47724) were purchased from Santa Cruz Biotechnology.