Anti phospho p53 ser15

Following the separation of proteins by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transfer to nitrocellulose membranes, the following antibodies were used: Anti-β-actin (Abcam; Ab8227), anti-HSP90 (Cell Signaling Technology, Frankfurt, Germany; No. 4874), anti-p53 (Santa Cruz Biotechnology, Heidelberg, Germany; sc-126), anti-phospho-p53 (Ser15) (Cell Signaling Technology; No. 9284), anti-phospho-p53 (Ser46) (Becton Dickinson; No. 558245), anti-MGMT (Sigma-Aldrich; HPA032136). Proteins were detected using the Odyssey 9120 Infrared Imaging System (Li-Cor Biosciences, Lincoln, Nebraska, USA). The membrane was dried at room temperature in the dark and scanned with Odyssey. Image J was used for the quantification.

Immunoblotting was performed following standard procedures as previously reported.⁵⁴ Cell homogenates were prepared by freeze–thawing and ultrasonication in a buffer containing detergents and protease inhibitors. 30 μg of cell proteins were denatured with Laemmli sample buffer, separated by electrophoresis on a 12.5% SDS-containing polyacrylamide gel and then electroblotted onto PVDF membrane (Carlo Erba reagents, Milan, Italy). The filter was first probed with the antibody specific for the protein of interest. The following primary antibodies were used: Rabbit polyclonal anti-PTEN (EX-BIO, Vestec, Czech Republic); rabbit polyclonal anti-LC3 (Sigma-Aldrich); rabbit polyclonal anti-phospho p53 (Ser15) (Cell Signaling technology, Danvers, Massachusetts, USA) and mouse monoclonal anti-p53 (Santa Cruz Biotechnology). The filter was subsequently stripped and re-probed with an antibody specific for β-tubulin (Sigma-Aldrich), as an index of homogenate protein loading in the lanes. Immunocomplexes were revealed by using a peroxidase-conjugated secondary antibody (Bio-Rad, California, USA), as appropriate, and subsequent peroxidase-induced chemiluminescence reaction (PerkinElmer, Massachusetts, USA). Densitometry of Western Blot bands was performed with the Quantity One-4.5.0 software (Bio-Rad) and with the free software Image J (1.48v; http://imagej.nih.gov/ij/).

Whole cell protein of untreated and 24 h Rapha Myr^® extract (0.5–1.25–2.5% v/v)-treated cells were prepared according to Laemmli (1970) and submitted to Western blot analysis, performed according to Grabowska et al., 2016 [72 (link)]. The primary antibodies used were: anti-integrin α5 (1:1000) (Immunological Sciences, Rome, Italy), anti-GAPDH (1:50,000) (Millipore, Darmstadt, Germany), anti-Poly (ADP-ribose)polymerase (PARP, 1:1000) (BD Biosciences, San Jose, CA, USA), anti-γH2AX Ser139 (1:1000) (Abcam, Cambridge, UK), anti-p53 (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p21 (1:500) (Sigma-Aldrich, St. Louis, MO, USA), anti-phospho-p53 Ser15 (1:250), anti-sirt 1 (1:250), anti-phospho-sirt 1 Ser47 (1:250), anti-sirt 3 (1:500), anti-sirt 5 (1:500), anti-sirt 6 (1:1000) and anti-sirt 7 (1:250) (Cell Signalling Technology, Denvers, CO, USA). Each protein target was detected by using specific secondary horseradish peroxidase-conjugated antibodies (1:2000) (Dako, Glostrup, Denmark) and an ECL system (Thermo Scientific, Rockford, IL, USA). The expression level of proteins was measured by densitometric analysis using the software Image J and GAPDH was chosen as the reference protein.

Proteins were extracted from each sample, at 5 days and 2 weeks after UVB exposure. Total protein (15 μg) was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted with anti-H2A histone family member X (H2AX), anti-γ-H2AX, anti-p53, anti-phospho-p53 (Ser15), anti-acetyl-p53 (Lys382), and anti-p21CIP1 antibodies (Cell Signaling Technology, Danvers, MA, USA), and with anti-TYR, anti-TYRP1, anti-TYRP2, and anti-GAPDH antibodies (Santa Cruz Biotechnology, Dallas, TX, USA), respectively.

HTR-8/SVneo cells (1 × 10⁶ cells per well) were plated onto six-well culture plates. After 48 h of sPIF treatment, cells were lysed in extraction buffer, as previously described.^{64 (link)} Twenty micrograms of total protein was resolved by SDS-PAGE (4–20% acrylamide) and transferred onto nitrocellulose membranes using a wet transfer method. After protein transfer, membranes were incubated at 4 °C overnight with rabbit polyclonal anti-phospho-p53 (Ser-15, 1 : 500 dilution, Cell Signaling Technology), mouse monoclonal anti-p53 (DO-1, 1 : 1000 dilution, Santa Cruz Biotechnologies, Inc.), anti-BCL2 (sc-509, 1 : 200 dilution, Santa Cruz Biotechnologies, Inc.), anti-BAX (sc-493, 1:200 dilution, Santa Cruz Biotechnologies, Inc.) or rabbit monoclonal anti β-actin (1 : 500 dilution, Sigma). After washing, the membranes were incubated with the peroxidase-coupled secondary antibody for 1 h at room temperature. Blots were developed using the SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Fisher Scientific Inc.).