Mdm2 ab 1

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).

Cells lysates were harvested by 2% SDS lysis buffer, heated and sonicated. The protein concentrations of the cell lysates were estimated using a Pierce^® BCA Protein Assay kit. The detailed protocol used for immunoblotting can be found in our previous report [9 (link)]. The primary and secondary antibodies used in the current study were listed as follows: p53 (DO-7) (#M7001, Dako, Glostrup, Denmark), MDM2 (Ab-1) (#OP46, Merck Millipore, Watford, UK), p21^WAF1 (EA10) (#OP64, Calbiochem (Millipore, Watford, Herts, UK), p-ERK (E-4) (#sc-7383, Santa Cruz, Dallas, TX, USA), ERK (K-23) (#sc-94, Santa Cruz), GAPDH (14C10) (#2118, Cell Signaling Technology, Danvers, MA, USA), WIP1 (F-10) (#sc-376257, Santa Cruz Biotechnology), phospho-p53 (S15) (ab1431, Abcam, Cambridge, UK), phospho-ATM(S1981) (#AF1655, R&D systems, Abingdon, UK), actin (#A4700, Sigma-Aldrich), secondary goat anti-mouse/rabbit horseradish peroxidase (HRP)-conjugated antibodies (#P0447/P0448, Dako).