Anti mouse igg or anti rabbit igg

Proteins were isolated by RIPA lysis buffer (Beyotime, Shanghai, China) and degenerated at 100℃ for 10min. In the next moment, proteins were separated by sodium dodecyl sulfatepolyacrylamide gels electrophoresis (BioRad, Berkeley, CA, USA) and transferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA). Blocking with 5% non-fat milk at 4℃ overnight and probing the membranes with polyclonal antibody overnight with the same conditions. After washed by TBST, the membranes were incubated with the anti-mouse IgG or anti-rabbit IgG (Abcam, Cambridge, MA) at room temperature for 1 hour. Primary antibodies were as follows: EZH2 (Abcam, USA), CDH2 (Abcam, USA), CDH4 (Abcam, USA), β-actin (Abcam, USA).

After nt-p65-TMD treatment in a dose-dependent manner for 2 h, RAW264.7 cells were lysed and the cell lysate was electrophoresed and transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories Inc., Hercules, CA, USA). The membrane was blocked using blocking buffer (4% bovine serum albumin in Tris-buffered saline containing Tween 20 (TBST) to prevent non-specific binding of antibodies. Blocked membrane was incubated with anti-DYKDDDDK (FLAG) or anti-β-actin antibody (Cell signaling, Beverly, MA, USA) at 4 °C overnight. After TBST wash, anti-mouse IgG or anti-rabbit IgG (Abcam, Cambridge, UK) was used to detect each primary antibody. For the chemiluminescence reaction, ECL reagent (Bio-Rad Laboratories Inc., Hercules, CA, USA) was added onto the membrane and the signal was observed using ChemiDoc (Bio-Rad Laboratories Inc., Hercules, CA, USA.).