Phosphorylated p53 serine 15 p p53

Following treatment with 2 and 4 µM bufotalin for 24 h, cells (1×10⁵ cells/ml) were harvested using a cell scraper at 4°C and the cell pellets were collected by centrifugation at a speed of 1,000 × g for 15 min at 4°C, and were lysed in RIPA cell lysis buffer on ice for 1 h. The total proteins were obtained from supernatant after centrifugation (16,099 × g) at 4°C for 30 min. The protein concentration of cell lysates was determined using a BCA assay kit (Sigma-Aldrich; Merck KGaA) as described previously (23 (link)). Equal amounts of protein (20 µg/lane) were separated on a 12% SDS-PAGE gel and transferred to polyvinylidene fluoride membranes. Following blocking with 5% non-fat milk at 4°C for 2 h, the membranes were incubated with primary antibodies p53 (cat. no. 9282; Cell Signaling Technology, Inc., Danvers, MA, USA) and phosphorylated p53 serine-15 (p-p53; cat. no. 9284, Cell Signaling Technology, Inc.) both at a dilution of 1:1,000 at 4°C overnight. Following washing with TBS solution, the membranes were incubated with corresponding secondary antibodies for 2 h at 4°C and visualized by Pierce enhanced chemiluminescence western blotting substrate (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA).

Levels of p53 (cat. no. 9282; Cell Signaling Technology, Inc.) and phosphorylated p53 serine-15 (p-p53; cat. no. 9284; Cell Signaling Technology, Inc.), both at a dilution of 1:1,000 in the treated cells were analyzed by immunofluorescence. Following treatment, 4×10⁴ cells were seeded, washed with PBS and fixed with 3.7% formaldehyde in PBS for 15 min at 4°C. Following rinsing with PBS, the cells were permeabilized with 0.2% Triton X-100 for 5 min at room temperature. The permeabilized cells were then blocked for 30 min with 0.1% bovine serum albumin (Gibco; Thermo Fisher Scientific, Inc.) at room temperature and incubated with p-p53 primary antibodies at 4°C overnight. Cells were then incubated with Alexa-488 labeled anti-rabbit immunoglobulin G (IgG) antibody (cat. no. 4412; 1:250; Cell Signaling Technology, Inc.) for 1 h at room temperature. The cells nuclei were stained with Hoechst 33342 (1 µg/ml) for 20 min at 37°C. A total of 3 fields of view of each slide were selected randomly and analyzed using a confocal fluorescence microscope (Zeiss LSM 510 Meta confocal microscope with LSM 510 software; Zeiss AG).