Prs4631

Cells and fresh tissues were lysed in 1×sodium dodecyl sulfate (SDS) lysis buffer. Equal amounts of total protein were separated by SDS-PAGE, electrotransferred onto polyvinylidene difluoride membranes (Pall Corporation, Mexico City, Mexico), and incubated overnight at 4°C with a SATB1 rabbit polyclonal antibody (1∶500 dilution, PRS4631; Sigma-Aldrich, St. Louis, MO, USA). Beta-actin (1∶10000, Sigma-Aldrich) was used as a loading control. Protein bands were evaluated by enhanced chemiluminescence (Thermo Fisher Scientific, RockFord, IL, USA).

Cells were cultured on glass coverslips, fixed with 4% paraformaldehyde, permeabilized using 0.1% Triton X-100, washed, and blocked with 5% bovine serum albumin. The cells were incubated with SATB1 primary antibody (1∶100, PRS4631, Sigma-Aldrich) followed by incubation with a rhodamine-conjugated secondary antibody (1∶50; Zhongshan Golden Bridge Biotechnology, Beijing, China). Coverslips were mounted with vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA) containing diamidino-2-phenylindole (DAPI) nuclear stain and examined under a fluorescence microscope, then the images were constructed using a software (LAS AF 2.2.1 version, TCSSP5, Leica, Germany).