Cip2a

Primary antibodies CHK1, pCHK1^Ser345, pCHK1^Ser317, CDC25C, pCDC25C^Ser216, Caspase 3 (#9662/#9664 (even mix)), and β‐actin were purchased from Cell Signaling (Beverly, MA, USA). pCDK1^Tyr15 (ab47594) were acquired from Abcam (Cambridge, UK). pH2A.X^Ser139 (#05‐636) was purchased from Millipore. Wee1 (sc‐5285), p53 (sc‐126), and Cyclin A (sc‐751) were obtained from Santa Cruz Biotechnology (Dallas, Tex, USA), whereas CIP2A was brought from Novus Biologicals (Littleton, Co, USA). Cells were harvested and then lysed in ice‐cold NP‐40 lysis buffer as previously described.24 Protein quantification was performed to ensure even loading by Bradford (Bio‐Rad Laboratories AB, Sundbyberg, Sweden) analysis. Proteins (15 μg protein/lane) were separated on Criterion TGX10% Midi Precast Gels in Tris/Glycine Buffers (Bio‐Rad, Hercules CA, USA) at 200 V for 40 minutes, and blotted on PVDF‐membrane using Bio‐Rad Transblot Turbo system according to the manufacturer's instructions. Membranes were blocked in 5% nonfat milk in TBST (20 mmol/L Tris‐Cl, 136 mmol/L NaCl [pH 7.6], 0.1% Tween 20) at room temperature for 1 hour, before they were probed with primary antibodies at 4°C overnight with gentle agitation. Secondary HRP‐conjugated antibodies were visualized using ECL‐plus reagent (GE Healthcare, Chalfont St Gils, UK) by exposure to X‐ray films.

Protein samples were analyzed as described [47 (link)]. Membranes were incubated with primary antibodies for pRb (554136), p130 (610261), Cdk1 (610038), E2F1 (all BD Biosciences, 554213); CIP2A (novus, NB100–68264); Cdk2 (sc-6248), Cdk4 (sc-260), Cdk6 (sc-177), cyclin A2 (sc-751), cyclin D1 (sc-718), c-Myc (sc-764), E2F2 (sc-633), E2F3 (sc-878), or GAPDH (all Santa Cruz Biotechnology, sc-25778); c-Myc (phosphor S62) (Abcam, ab51156 )

Immunoblot analysis was performed as previously described [10 (link)]. The following proteins were evaluated by immunoblot: CIP2A (1:500; Novus Biologicals, CO, USA), pERK (1:1000; Cell Signaling Technology, Boston, MA, USA), ERK (1:5000; Zymed, Grand Island, NY, USA) and β-actin (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA). β-actin was used as a loading control. Immunoblot quantification was performed using Image J software (http://rsb.info.nih.gov/ij/index.html).