Supersignal west pico enhanced chemiluminescence reagent

A 20-mm section of injured spinal cord tissue, centered on the epicenter of the injury, was lyzed in RIPA + PMSF (at a 100:1 ratio of RIPA:PMSF) buffer on ice. The collected proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a PVDF membrane, and incubated overnight with primary antibodies at 4 °C (Map-2, 1:2000, Abcam, UK; GFAP, 1:2000, Abcam, UK). This was followed by incubation with a secondary antibody (Santa Cruz Biotechnology; 1:2000 in blocking solution) for 1 h at room temperature. The blots were then visualized using the SuperSignal West Pico enhanced chemiluminescence reagent (Thermo Scientific) and quantified using Image J software.

The following antibodies were used in this study at the indicated dilutions: anti-CIP2A (CST #14805; 1 : 1000), anti-GAPDH (Sigma-Aldrich G9545; 1 : 20 000), anti-GFP (gift from Laurence Pelletier, 1 : 10 000), anti-KAP1 (Bethyl A300-274A, 1 : 5000), anti-POLE3 (Bethyl A301-245A-1; 1 : 2000), anti-POLE4 (Abcam ab220695; 1 : 200), anti-Tubulin (Millipore CP06, 1 : 2000), anti-pCHK1 (S345) (Cell Signaling #2348, 1 : 1000), anti-CHK1 (Santa Cruz sc8408, 1 : 500), anti-pRPA32 (S33) (Bethyl A300-246A-3, 1 : 20 000), anti-RPA32 (Abcam ab2175, 1 : 500). The following secondary antibodies for immunoblotting were used in this study: peroxidase-conjugated AffiniPure Bovine Anti-Goat IgG (Jackson Immuno Research 805-035-180) and peroxidase-conjugated Sheep Anti Mouse IgG (GE Healthcare NA931 V). All peroxidase-conjugated secondary antibodies were used at a dilution of 1 : 5000. Protein bands were detected using the SuperSignal West Pico enhanced chemiluminescence reagent (Thermo Fisher Scientific). The following siRNAs from Dharmacon were used in this study: control, siGENOME Non-targeting Pool #2 (D-001206-14-05); POLE3, siGENOME SMARTpool (M-008460-01-0005); POLE4, siGENOME SMARTpool (M-009850-01-0005); APEX2, siGENOME SMARTpool (M-013730-00-0005). ATR inhibitors VE-821 and AZD6738 were purchased from SelleckChem.

After a PBS wash, the cells were extracted in cold lysis buffer (20 mM Tris HCl, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM EDTA, 1.0 mM sodium orthovanadate, 50 mM sodium fluoride, 200 mM phenylmethanesulfonyl fluoride, and 40 ml/ml of protease inhibitor cocktail). The proteins were separated by 12% acrylamide gel electrophoresis and transferred to a PVDF membrane. The member was blocked for 1 h in 10% skim milk at room temperature. The membranes were incubated overnight at 4°C with antibodies to Map-2 (1:2000, Abcam), GalC (1:2000, Abcam), Samd1 (1:1000, Signaling Technology, Danvers, MA, U.S.A.), and Smad5 (1:1000, Cell Signaling Technology, Danvers, MA, U.S.A.). Next, the blots were washed and incubated with a secondary antibody (Santa Cruz Biotechnology; 1:2000 in blocking solution) for 1 h at room temperature. After being washed three more times with TBS-T, the blots were developed using SuperSignal West Pico enhanced chemiluminescence reagent (Thermo Scientific).