Mouse anti dnmt1

Total protein was extracted from N134 cells infected with RCASBP-Dnmt1 or RCASBP-control using a RIPA lysis buffer (Pierce, Rockford, IL). The extracted proteins were quantified and equal amounts of protein were separated by SDS-PAGE for western blotting. The blots were incubated overnight at 4 °C with primary antibodies: mouse anti-GAPDH (1:1000 dilution) and mouse anti-Dnmt1 (1: 2000 dilution) (Abcam, Cambridge, MA), respectively. The secondary antibody used was: 1:5000 dilution of anti-mouse IgG-HRP (Thermo Fisher Scientific, Rockford, IL). The small differences in loading were corrected by comparison with the loading control, GAPDH.

IF staining was used to evaluate the expression levels of Dnmt1 and Ki67 in pancreatic tissues from treated mice. The sections were incubated overnight at 4 °C with primary antibodies: pig anti-insulin antibody (1:500 dilution; DAKO, CA, USA), mouse anti-Dnmt1 (1:200 dilution), and mouse anti-ki67 (1:1000 dilution) (Abcam, Cambridge, MA), respectively. After washing, the slides were incubated with goat anti-mouse Alexa Fluor 488 (Life Technologies, Grand Island, NY) and goat anti-guinea pig Alexa Fluor 647 (Life Technologies, Grand Island, NY) (both 1:200 dilutions) for 45 min in the dark. The slides were assembled in Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA). Images were obtained with an epifluorescence microscope with an attached camera.