Laemmli denaturation buffer

Cells transfected with pre-miR-222 or pre-miR-NC for 48 hours were harvested and washed using ice cold PBS, centrifuged and the supernatant discarded. The pellet was solubilized in RIPA lysis buffer (Pierce) containing protease and phosphatase inhibitor mixture (Roche). Cell lysates were placed on ice for 30 minutes and centrifuged for 10 minutes at 12,000 rpm at 4°C. The protein concentration was measured using the Bio-Rad Protein Assay (Bio-Rad). Protein samples were mixed at 1:1 ratio with Laemmli denaturation buffer (Bio-Rad) and β-mercaptoethanol (Sigma Aldrich) at a final dilution of 1/40 and boiled for 10 minutes at 95°C. Approximately 25 μg of protein was loaded and fractionated using a 10% SDS-PAGE gel (Bio-Rad). The protein was transferred onto a nitrocellulose membrane (Bio-Rad) and immunoblotted with antibodies against cleaved PARP (Cell Signaling Technology) and β-actin (Sigma Aldrich) as a loading control. Secondary antibody was anti-mouse HRP-linked (Cell Signaling Technology). Visualization of the proteins was done using the ChemiDoc-It Imaging System (UVP).

Neuroblastoma cells treated with palbociclib (Selleck Chemicals) for 24 h were harvested and washed using ice-cold PBS, centrifuged and the supernatant discarded. The pellet was solubilized in RIPA lysis buffer (Pierce) containing protease and phosphatase inhibitor mixture (Roche). Cell lysates were placed on ice for 30 min and centrifuged for 10 min at 12,000 rpm at 4°C. The protein concentration was measured using the Bio-Rad Protein Assay (Bio-Rad). Protein samples were mixed at 1:1 ratio with Laemmli denaturation buffer (Bio-Rad) and β-mercaptoethanol (Sigma Aldrich) at a final dilution of 1/40 and boiled for 10 min at 95°C. Approximately 25 µg of protein was loaded and fractionated using a 10% SDS-PAGE gel (Bio-Rad). The protein was transferred onto a nitrocellulose membrane (Bio-Rad) and immunoblotted with rabbit monoclonal antibody against phospho-Rb (Ser⁷⁸⁰) (Cell Signaling Technology), rabbit polyclonal antibody against total Rb (Abcam), and mouse monoclonal antibody against β-actin (Sigma Aldrich) as a loading control. Secondary antibody was anti-mouse HRP-linked (Cell Signaling Technology) and anti-rabbit HRP-linked (Cell Signaling Technology). Visualization of the proteins was done using ChemiDoc-It² Imaging System (UVP, Sopachem).