Ab76009

Cells were lysed in the lysis buffer and the protein concentration was measured using the BCA protein assay kit (Thermo Fisher Scientific). The 10 μg proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto the polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were blocked with 5% skim milk for 2 h and incubated with rabbit anti-MYBL2 (1:1000, ab76009, Abcam, Cambridge, UK), cyclin A2 (1:10000, ab32386, Abcam, Cambridge, UK), cyclin B1 (1:20000, ab32053). Abcam, Cambridge, UK), GAPDH (1:10000, ab180630, Abcam, UK) and Plk1 (1:1000, ab155095, Abcam, Cambridge, UK) overnight at 4°C. Then the membranes were cultured with secondary antibody goat anti-rabbit IgG H&L (ab205718, Abcam, Cambridge, UK) for 1 h at room temperature. Finally, the electrochemiluminescence kit (ECL; Pierce Biotechnology) was used for protein development.

Immunostaining of paraffin-embedded xenograft tumor tissues was performed. IHC was performed by means of streptavidin coupled with peroxidase, with rabbit anti-MYBL2 antibody (1:100, ab76009, Abcam, Cambridge, UK) and Ki67 antibody (1:50, ab15580, Abcam, Cambridge, UK) as primary antibodies and goat anti-rabbit IgG H&L (ab205718, Abcam, Cambridge, UK) as secondary antibody. Non-immune serum was used to replace primary antibody as negative control. Sections were observed under a 400× or 200× microscope (ZEISS, Germany).

The fresh paired cancer and para-cancer normal tissues, and formalin-fixed, paraffin-embedded (FFPE) cancer tissues of clinical CRC patients were obtained from the Second Affiliated Hospital of Zhejiang University School of Medicine. The collection and using of the samples were performed according to the ethical standards formulated in the Declaration of Helsinki. Written informed consent was obtained from each patient, and the study was approved by the ethics committee of the Zhejiang University School of Medicine, China. The clinicopathological characteristics of the clinical specimens are shown in Table S1 and S2.
The IHC staining was performed with Envision detection system (Dako, Denmark) in FFPE CRC tissues. The primary antibodies included anti-RRM2 (ab57653, abcam) and anti-MYBL2-pT487(ab76009, abcam) [39 (link)]. The IHC score for slides were determined as previously described [40 (link), 41 (link)].