Secondary goat anti mouse rabbit horseradish peroxidase conjugated antibodies

1 × 10⁶ cells/mL (5 × 10⁶ cells/mL for CLL primary cells or healthy PBMCs) were seeded in 2 mL per well of a 6-well plate (Corning) and exposed to a range of concentrations of E7107 and/or idasanutlin. Concentrations are indicated in the figure legends. Protein lysates were harvested using 2% SDS lysis buffer at 24 h, heated at 95 °C for 10 min, and sonicated. Protein concentration was measured using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, UK). Primary antibodies against p53 (DO-7) (#M7001, Dako), MDM2 (Ab-1) (#OP46, Merck Millipore), p21^WAF1 (Calbiochem), PARP (Trevigen), SF3B1 (Sigma), Actin (Sigma), Phospho-Rb (Ser780) (Cell Signalling), Phospho-Rb (Ser807/811) (Cell Signalling), Phospho-Rb (T821) (Abcam) and secondary goat anti-mouse/rabbit horseradish peroxidase-conjugated antibodies (Dako) were used. All antibodies were diluted in 5% (w/v) nonfat milk or BSA in TBS-tween20. Proteins were visualized using enhanced chemiluminescence reagents (GE Healthcare).

The 1 × 10⁶ cells/mL (5 × 10⁶ cells/mL for primary CLL cells) were seeded in 2 mL per well of a 6-well plate (Corning) and subjected to the corresponding manipulation (siRNA transfection and/or exposure to siremadlin or idasanutlin). Protein lysates were harvested using 2% SDS lysis buffer at 24 h, heated at 95 °C for 10 min, and sonicated. Protein concentration was measured using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Primary antibodies against p53 (DO-7) (#M7001, Dako), p21^WAF1 (Calbiochem), SF3B1 (Sigma), MDM2 (Ab-1) (#OP46, Merck Millipore), Actin (Sigma) and secondary goat anti-mouse/rabbit horseradish peroxidase-conjugated antibodies (Dako) were used. All antibodies were diluted in 5% (w/v) non-fat milk or BSA in TBS-tween20. Proteins were visualized using enhanced chemiluminescence reagents (GE Healthcare, Chicago, IL, USA).