Uvchem detection device

Protein lysates were extracted from cells treated with 1 μM RITA for 24, 48, and 72 h, lysed 30 min on ice in radioimmunoprecipitation assay (RIPA) Buffer supplemented with Complete Protease Inhibitor Cocktail Tablets and PhosSTOP Phosphatase Inhibitor Cocktail Tablets (Roche). 50 μg proteins were separated on 10% SDS-PAGE then transferred to Amersham-Hybond™-C Extra (GE Healthcare) membranes. Transferred membranes were blocked in 5% milk powder in Tris-buffered saline and Tween 20 and then incubated for 12 to 24 h, using the following antibodies and dilutions: CDKN1A (1:500, cat ab-7960, Abcam), TP53 (1:500, cat#sc-71817, Santa Cruz Biotechnology), MDM2 (1:1000, IF2, cat#33-7100, Life Technologies), beta-actin (1:2000, cat#A5541, Sigma-Aldrich), GAPDH (1:2000, cat#MAB374, Merck Millipore), and HRP-conjugated anti-rabbit IgG (1:2000; GE Healthcare) or HRP-conjugated anti-mouse IgG (1:2000; GE Healtcare) was added for 1 h at room temperature. Proteins were visualized using Amersham ECL Plus™ western blotting detection reagents (GE Healthcare) and the UVchem Detection Device (Biometra).

Total proteins were isolated from cells using RIPA buffer (Beyotime Biotechnology, Shanghai, China), separated by dodecyl sulfate, sodium salt-Polyacrylamide gel electrophoresis (SDS-PAGE, 10%), and transferred onto polyvinylidene didluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked by 5% skimmed milk at room temperature for 1 h, followed by incubation with primary antibodies (1: 1,000) at 4°C overnight. After washing with a TBST (Tris-buffered saline containing 0.1% Tween-20) buffer three times, the membranes were incubated with HRP-linked secondary antibodies (1:2,000) at room temperature for 1 h and then washed with a TBST buffer for five times. The proteins were visualized by Amersham ECL Plus (Amersham, Arlington Heights, IL, USA) and detected with UVchem Detection Device (Biometra, Gottingen, Germany). Primary antibodies against KDM1A, ZNF346, or β-actin (Abcam, Cambridge, MA, USA) were used in this study.