Cells were allowed to recover overnight in Cunningham me- dium at 26 °C without any drug selection. In the morning, the transfected cells were diluted to the concentration of 2×105 cells ml−1 with fresh Cunningham medium containing the respective selection antibiotic. The cells were evenly...
The microdissected tissues were placed in individual polymerase chain reaction (PCR) tubes and digested overnight at 50°C in 10 μl of lysis buffer containing 1 mg/ml proteinase K (Boehringer Mannheim, Indianapolis, IN), 10 mmol/L Tris/HCL (pH 8), 50 mmol/L KCl, 0.45% NP40, and ...
Anti-pRb, anti-Rb, anti-cyclin D1, anti-Cdk4 (1:1000), and anti-actin (1:400) antibodies were added overnight at 4°C. All antibodies were purchased from Cell Singling Technology, 9308, 9309, 2926, 2906, and 8456 respectively. The horseradish peroxidase-conjugated goat anti-rabbit ...