Anti phospho prb ser807 811

Western blot analyses were performed as previously described [50 , 51 ]. Briefly, the cells were washed twice with PBS and lysed via sonication in lysis buffer (50 mM Tris-HCl, pH 7.5; 1% NP-40; 1 mM Na3VO4; 150 mM NaCl; 20 mM Na4P2O7; 100 mM NaF; 1% Na-deoxycholate; 0.1% SDS; 1 mM EDTA; phosphatase inhibitor cocktail (Sigma) and protease inhibitor cocktail (Sigma)). The samples were separated on 6–15% SDS-PAGE gels, transferred to nitrocellulose membranes (Protran BA83, Whatman) and immunostained. The following primary antibodies and dilutions were used: Anti-p21 [C-19] 1:200 (Santa Cruz, #sc-397), anti-p53 [FL-393] 1:200 (Santa Cruz, #sc-6243), Anti-p16 (M-156) 1:200 (Santa Cruz, #sc-1207), anti-pRb 1:500 (BD-Pharmingen), anti-phospho-pRb (Ser807/811) 1:1000 (Cell Signaling), anti-phospho-histone H2A.X (Ser139) 1:1000 (Millipore 05–636) and monoclonal anti-α-tubulin 1:1000 (Sigma 9026). Horseradish peroxidase-labeled rabbit anti-mouse (Amersham, diluted 1:3000) and goat anti-rabbit (Abcam, #6721, diluted 1:3000) secondary antibodies were used. The proteins were visualized using an ECL detection system (Amersham Biosciences).

In situ proximity ligation assay (PLA) was performed to assess protein-protein interaction [53 (link), 54 (link)]. Cells grown on 8-well culture slides (Labtek chamber slides (Nunc, #154534, Thermo Fisher) were fixed and subjected to in situ PLA using the Duolink Detection kit (Olink Bioscience, Sweden) according to the manufacturer's instructions. Briefly, melanoma tissue sections and fixed cell culture slides were incubated after blocking, with antibodies directed against pRB (Cell signaling, France), anti-phospho-pRb (Ser807-811) (Cell signaling, France) and E2F1 (Santa Cruz, France). Slides were thereafter incubated with PLA minus and PLA plus probes containing the secondary antibodies conjugated with oligonucleotides. Circularization and ligation of the oligonucleotides was followed by an amplification step. The products were detected by a complementary fluorescently labeled probe. Protein complexes were visualized using a laser-scanning confocal microscope (Leica-Lasertechnik) as bright fluorescent signals.