Bs 4056r

For immunohistochemical (IHC) staining, formaldehyde-fixed, paraffin-embedded sections of tumor tissues were obtained from mouse models. Briefly, the deparaffinized sections were then treated with methanol containing 3% hydrogen peroxide for 15 min. The sections were washed with PBS and blocked with blocking serum for 30 min at room temperature. The sections were then incubated with primary antibodies, including anti-SMURF2 (bs-4056R, Bioss), anti-ID2 (MA5-32891, Invitrogen), and anti-p21 (ab107099, Abcam) at 4°C overnight. The following day, the primary antibodies were washed off and sections were incubated with HRP-conjugated secondary antibodies for 2 h at room temperature. The nuclei were stained with hematoxylin, and DAB substrate (ZLI-9019, ZSGB-Bio, Beijing, China) was added to detect the proteins. The obtained immunohistochemistry images were analyzed with following scoring criteria. Cells with 0% staining were scored as 0; cells with 1%-33% staining were scored as 1; cells with 34%-66% staining were scored as 2; cells with 67%-100% staining were scored as 3. Additionally, the staining intensities were evaluated into four grades: 3 (strong), 2 (moderate), 1 (weak) and 0 (none). The final score was defined as the score of percentage classifications multiplied by intensity grades.

Cycloheximide (CHX) and MG132 were purchased from MCE (HY-12320; MedChemExpress, China) and Selleck (S2619, China), respectively. The antibodies used in this study were as follows: anti-β-actin (M1210-2; HUABio, Hangzhou, China), anti-SMURF2 (12024S; CST, Danvers, Massachusetts, USA; bs-4056R; Bioss, Beijing, China), anti-ID2 (ab166708; Abcam, China; 3431S; CST; MA5-32891; Invitrogen, USA), anti-E2A (ab228699; Abcam), anti-Flag (14793S; CST), anti-GFP (OSE00003G; Invitrogen), anti-HA (3724T; CST), anti-Rabit IgG (3900S; CST), anti-p21 (10355-1-ap; Proteintech, Wuhan, Hubei, China; ab107099; Abcam; 2947T; CST), anti-Ubiquitin (3936T; CST).