Anti cbx4

Centrifuged cells or striatum from mice were lysed in radioimmunoprecipitation assay (RIPA) buffer with the addition of protease inhibitor cocktail (Sigma-Aldrich, Cat. No. P83490) buffer, briefly sonicated, and boiled for 5 min at 95°C. 30 μg protein extracts were resolved by 4%–12% SDS-PAGE gels and transferred to 0.2 μM nitrocellulose membrane (Amersham, Cat. No. GEH10600001) for 2 h at 35 V. Membranes were blocked with 5% non-fat dry milk in PBS/0.1% Tween 20 and incubated with the following indicated primary and secondary antibodies: anti-β-actin 1:20,000 (Sigma, Cat. No. A2066), anti-GDNF 1:500 (Santa Cruz Biotechnology, Cat. No. sc-328), anti-TH 1:1,000 (Millipore, Cat. No. AB152), anti-DAD1 1:1,000 (Novus Biologicals, Cat. No. NB600-669), anti-LRRC14 1:1,000 (Novus Biologicals, Cat. No. NBP1-80049), and anti-CBX4 1:500 (Santa Cruz Biotechnology, Cat. No. sc-517216). The antibody against GDNF detects the mature form of GDNF, with an apparent molecular weight of approximately 17 kDa. Proteins of interest were visualized with the Amersham ECL Detection Reagents (GE Healthcare by Sigma, Cat. No. RPN2105). Western blotting images were acquired with Image Quant LAS 4000mini system, and band intensity was calculated using Image Quant TL software.