Primary antibodies against p65

Immunofluorescence analysis was performed as previously described (Yu et al., 2019). Briefly, AN3-CA cells (transfected with pcDNA or a pcDNA-based NDRG1-overexpression vector) or RL95-2 cells (transduced with a control shRNA or an shRNA against NDRG1) were seeded onto coverslips in plates. On the following day, the cells were fixed with 4% paraformaldehyde (Sigma Aldrich, St. Louis, MO, USA) and permeabilized with 0.3% Triton X-100 (Sigma Aldrich, St. Louis, MO, USA). The cells were then blocked with 1% bovine serum albumin solution in phosphate-buffered saline and probed overnight with primary antibodies against p65, ZEB1, and E-cadherin (all from Cell Signaling Technology, Danvers, MA, USA). Following this, the cells were probed with secondary antibodies conjugated with Alexa Fluor 594 or Alexa Fluor 488 (Invitrogen, Waltham, MA, USA). Nuclei were stained with DAPI (Invitrogen, Waltham, MA, USA). A negative control was used to confirm the specificity of the antibodies used in this study (Supplementary Figure S1). Images were examined and captured using a confocal microscope (LSM710; Carl Zeiss, Oberkochen, Germany).

Experiments were carried out as previously described^{54 (link)}. Nuclear and cytoplasmic extracts were isolated using nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific). Cell lysates were electrophoresed in 4–12% gradient polyacrylamide gels (Invitrogen), and the resolved proteins were transferred to nitrocellulose membranes (Amersham Biosciences, Arlington Heights, IL, USA). Membranes were blocked with blocking buffer (Takara Bio), washed in Tris-buffered saline with Tween (TBST), and incubated with primary antibodies against p65 (Cell Signaling Technology) and Lamin B1 (Abcam) diluted 1:1000 in Can Get Signal Immunoreaction Enhancer Solution 1 (TOYOBO, Osaka, Japan). Prior to hybridisation with primary antibodies, membranes were cut at the each expected blots point. After washing in TBST, secondary anti–rabbit IgG antibodies (Cell Signaling Technology) were added. Immunoreactivity was detected with ECL Prime (Amersham Biosciences) and photographed on an Ez Capture MG (ATTO, Tokyo, Japan). Band densities were calculated using CS Analyzer 3.0 (ATTO).