Rabbit anti p53 monoclonal antibody

HTR-8 cells were cultured on poly-L-lysine–coated coverslips in 24-well plates, treated with control vector (vector) or lentiviruses carrying short hairpin RNA (shTTP), cultured for 48 h, washed three times with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde–PBS for 10 minutes, and stained with primary antibodies [rabbit anti-STMN1 monoclonal antibody (1:150; Abcam) and mouse anti-TTP monoclonal antibody (1:100; Santa Cruz Biotechnology)] overnight at 4°C. The next day, secondary fluorescent Alexa Fluor 488 donkey anti-rabbit IgG (H+L) and Alexa Fluor 594 donkey anti-mouse IgG (H+L) antibodies (Life Technologies) were used after the plates had been washed three times with PBS. Nuclei were counterstained using 4,6-diamidino-2-phenylindole (DAPI; Abcam). Coverslips were mounted on the glass slides and the slides were visualized using a Leica microscope (Wetzlar, Germany). For the chorionic tissues, rabbit anti-p53 monoclonal antibody (1:1600; Cell Signaling Technology) was used as primary antibody.

Total protein was extracted by TRIzol^TM reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer and quantified by the Bradford protein assay using the Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad, Hercules, CA, USA). Approximately, 20 µg of the extracted proteins were separated by 12% SDS-PAGE. The separated proteins were transferred to PVDF membranes Thermo Scientific, Waltham, MA, USA) at 100 V/250 A for 45 min. using the TE77XP Semi-Dry Transfer Units (Hoefer, Holliston, CA, USA). The membranes were then incubated overnight with rabbit anti-p53 monoclonal antibody (Cell signaling technology, Danvers, MA, USA) at 4 °C. After washing with PBS-T buffer (1× PBS buffer with 0.05% Tween-20), the membrane was exposed to the horseradish peroxidase linked goat anti-rabbit monoclonal antibody (Cell signaling technology, Danvers, MA, USA) at room temperature for 1 h. using ImageQuant™ LAS 4000 (GE Healthcare, Piscataway, NJ, USA). The immunoreactive signal was visualized using Amersham™ ECL™ Primers and Western Blotting Detection Reagents (GE Healthcare, Piscataway, NJ, USA). β-actin was used as a control.