Anti dnmt1

Paclitaxel was purchased from Bristol-Myers Squibb Pharmaceutical Ltd. MG-132(#S2619) was purchased from Sigma. The antibodies used for Western blotting included anti-UbC13 (#ab25885, Abcam), anti-DNMT1 (#24206-1-AP, Proteintech), anti-CHFR (#6904, Cell Signaling Technology), anti-Aurora A (#12100, Cell Signaling Technology), anti-Ub (#3933, Cell Signaling Technology), anti-HA(#ab003, Lianke), anti-EGFP (#ab006, Lianke), anti-GAPDH (#Mab5465, Lianke), anti-mouse HRP (#7076, Cell Signaling Technology), and anti-rabbit HRP (#7074, Cell Signaling Technology). Antibody anti-DNMT1 (#ab13537, Abcam) was used for immunoprecipitation.

IHC was performed following a previously described method [30 (link)]. The following antibodies were used: anti-CD34, anti-VEGF, anti-DNMT1, anti-TRAF1, and anti-NF-κB p65 (Proteintech, Wuhan, China). Staining intensity was scored manually by two independent experienced pathologists as follows: 0 = no staining, 1 = weak staining, 2 = moderate staining, and 3 = strong staining. The percentage of positive cells was also assessed according to four scores: 1 (0-10%), 2 (11-50%), 3 (51-80%), and 4 (81-100%). The final IHC score was calculated by multiplying the intensity score by the percentage of positive cells. CD34 antibody was used to stain vascular endothelial cells, and then the microvessel density (MVD) was calculated. The field of maximal CD34 expression was found in tumor tissues. Within this field, the area of maximal angiogenesis was selected, and microvessels were counted at 200× magnification. Low MVD was indicated by scores from 0 to 3. High MVD was indicated by scores ≥4 [31 (link)].

Western blot analysis was conducted as previously described.^{14 (link)} The following primary immunoblotting antibodies were used: anti-GAPDH, anti-E-cadherin, anti-Fibronectin, anti-Vimentin, anti-Snail, anti-AKT2, anti-p-AKT, anti-Rb, anti-p-Rb (Epitomics, Burlingame, CA, USA), anti-E2F1, anti-CDK4 and anti-DNMT1 (Proteintech, Chicago, IL, USA), anti-N-cadherin, anti-p27, anti-p21, anti-c-myc, anti-ERBB3, and anti-CCND1 (Cell Signaling Technology, Beverly, MA, USA).