Rabbit anti p21 monoclonal antibody

Roswell Park Memorial Institute (RPMI) 1640 medium was purchased from Gibco/Thermo Fisher Scientific, Inc. Fetal bovine serum was obtained from Biological Industries. Mouse anti-p53 monoclonal antibody (catalog no. 2524S), rabbit anti-phospho-p53 polyclonal antibody (catalog no. 2521S), and rabbit anti-p21 monoclonal antibody (catalog no. 2947S) were purchased from Cell Signaling Technology, Inc. Rabbit anti-DLGAP5 polyclonal antibody (catalog no. PA5-82197) was purchased from Invitrogen Company. Mouse anti-actin monoclonal antibody (catalog no. sc-47778) was purchased from Santa Company. Every assay was done in triplicate.

To analyse the intracellular distribution of p21 in LECs, cells were seeded on glass coverslips in 24-well plates. The cells were grown in endothelial cell growth medium supplemented with 10% human serum for 24 h, fixed with methanol at −20°C and permeabilized with 0,1% TritonX-100 for 5 min at room temperature. Permeabilized cells were rinsed three times with PBS and incubated in blocking solution (1% bovine serum albumin/PBS) for 30 min at room temperature to remove nonspecific binding of the antibody. p21 was detected using a rabbit monoclonal anti-p21 antibody (#2947, Cell Signaling, Danvers, MA, USA) and an Alexa Fluor 546 goat anti-rabbit IgG secondary antibody. The slides were mounted in Aqua-Poly/Mount (Polysciences, Inc., Warrington, PA, USA) and viewed with fluorescence microscopy. Cells were counterstained for nuclei with Hoechst staining.