Rabbit anti p27 sc 528

Cortical tissues were treated with tissue lysate buffer (Pro-prep^TM; Intron Inc., Gyeonggi-do, Korea). Protein concentration was measured by the Bradford assay (Bio-Rad, Hercules, CA, USA). The protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. Western blot analysis was performed using the following antibodies: rabbit anti-4-HNE (ab46545; Abcam), mouse anti-3-NT (ab7048; Abcam), mouse anti-p53 (#OP09; Calbiochem, San Diego, CA, USA), rabbit anti-p27 (sc-528; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-p21 (#2947; Cell Signaling Technology, Danvers, MA, USA), mouse anti-γH2AX (#05-636; EMD Millipore, Billerica, MA, USA), rabbit anti-Bax (#2772; Cell Signaling Technology), rabbit anti-Bcl-2 (#2876; Cell Signaling Technology), rabbit anti-cleaved caspase-3 (#9661; Cell Signaling Technology), rabbit anti-cleaved PARP-1 (#9542; Cell Signaling Technology), goat anti-GFAP (ab53554; Abcam), rabbit anti-Iba-1 (#016-20001; Wako), and mouse anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA). The next day, the membranes were washed with 0.1% Tween-20 in phosphate-buffered saline (PBS) and incubated with each biotinylated secondary antibody.

Preparation of lysates and immunoblot analyses were performed as described previously^{45 (link)} using Tris lysis buffer (50 mM Tris–HCl pH 7.8, 150 mM NaCl, 1% IGEPAL CA-630) containing 100 mM NaF, 100 mM β-glycerophosphate, 20 μg/ml RNase A, 20 μg/ml DNase and 1/300 protease inhibitor cocktail (P8340, Sigma–Aldrich) and 1/100 phosphatase inhibitor cocktail #2 (P5726, Sigma–Aldrich). Antibodies used in this study were purchased from the following sources: rabbit anti-Cdk2 (M2 SC-163, Santa Cruz Biotechnology), mouse anti-tubulin (Developmental Studies Hybridoma Bank, University of Iowa), mouse anti-Cyclin A (SC-751, Santa Cruz Biotechnology), mouse anti-cyclin E (SC-198, Santa Cruz Biotechnology), rabbit anti-CDK4 (sc-260, Santa Cruz Biotechnology), mouse anti-Rb (G3-245, BD Pharmingen), rabbit anti-p27 (sc-528, Santa Cruz Biotechnology) and anti-HA High affinity (3F10, 11867423001, Sigma-Aldrich). Secondary antibodies used for western blot analysis were goat anti-mouse (31430, Thermo Scientific) and, goat anti-rabbit (31460, Thermo Scientific). mouse anti-tubulin hybridoma cell line (clone #12G10) was developed by J. Frankel and E.M. Nelson under the auspices of the NICHD and maintained by the Developmental Studies Hybridoma Bank.