Duolink in situ proximity ligation assay for protein: protein interactions Duolink proximity ligation assay kit composed of anti-rabbit PLA probe plus, anti-mouse PLA probe minus and detection kit 613 was purchased from Olink Bioscience. was transformed using 5 l of the ligation mixture, and bacterial colonies bearing the insert were selected on LB-agar plates containing 50 g/ml of kanamycin. Plasmid DNAs were purified from individual clones using a QIAprep spin miniprep kit (Qiagen) and the presence of the expected insert was confirmed by sequencing. 2.3. Transient transfection HEK 293 cells were transiently co-transfected with CFP and YFP tagged constructs using FuGENE HD transfection reagent (Roche Applied Science). Transient knockdown of cellular RPS3 was achieved by transfecting with RPS3 specific siRNA (Dharmacon) using Dharmafect 1 transfection reagent (Dharmacon) as previously described [14]. 2.4. Fluorescence resonance energy transfer (FRET) analysis by laser scanning confocal microscopy Cells co-transfected with CFP and YFP constructs were fixed in 10% neutral buffered formalin and washed in PBS before being mounted onto slides using Vectashield mounting media. Using acceptor photobleaching method [15], protein:protein interactions were analyzed by using a Zeiss laser scanning confocal microscope (LSM 510 Meta). FRET efficiency (E) was calculated using the equation E=1?(IDA/ID), where IDA and ID represent steady state CFP fluorescence in the presence and absence of the YFP, respectively. FRET efficiency was determined for a minimum of 50 cells of same fluorescence intensity and used for statistical manipulations. 2.5. Antibodies Custom synthesized rabbit monoclonal RPS3 antibody (Proteintech) was used for immunoblotting and immunofluorescence. Anti-p53 antibody (DO-1) was purchased from Santa Cruz Biotechnology. The mouse monoclonal cocktail prepared from IF2, 4B11 PF-06380101 and 2A10 antibodies from EMD Biosciences was used for detecting MDM2 by immunoblotting. MDM2 antibody, clone IF2 was used for immunofluorescence applications. Anti-glyceraldehyde 3-phosphate dehydrogenase antibody (GAPDH) was purchased from Chemicon. 2.6. Duolink in situ proximity ligation assay for protein: protein interactions Duolink proximity ligation assay kit composed of anti-rabbit PLA probe plus, anti-mouse PLA probe minus and detection kit 613 was purchased from Olink Bioscience. Formalin fixed cells were permeabilized using 0.1% triton-X100 and blocked overnight at 4 C in 1% BSA. Primary antibody mixtures were prepared in the blocking solution by adding RPS3 (1:200) to p53 (DO1, 1:100) or MDM2 (IF2, 1:200) antibodies and cells were incubated with the mixture for 1 h at room temperature. All subsequent incubations were performed in a humidifying chamber maintained at 37 C. PLA probes were diluted in blocking solution and PF-06380101 all other Duolink reagents were diluted according to the manufacturers instructions. After 90 min incubation with the PLA probes, cells were washed in PBS and incubated with the hybridization mixture for 15 min and ligation mixture for an additional 15 min with a TBS-T (10 mM Tris [pH 7.5], 150 mM Nacl and 0.1% Tween 20) wash in between. After washing in TBS-T, cells were incubated with the amplification mixture for 90 min followed by the detection mixture for 1 h. The cells were then washed in 2 SSC, 1 SSC, 0.2 SSC, 0.02 SSC followed by 70% ethanol wash. Samples were air dried and mounted with Olink mounting media containing DAPI nuclear stain. Detection of the interaction signals was carried out by fluorescence microscopy Rabbit polyclonal to CaMK2 alpha-beta-delta.CaMK2-alpha a protein kinase of the CAMK2 family.A prominent kinase in the central nervous system that may function in long-term potentiation and neurotransmitter release. using Zeiss Axioplan 2 upright microscope equipped with Photometrics Coolsnap HQ CCD camera. The filter sets used for visualizing the fluorescent signals include DAPI (EX 360/40, EM 460/50) and Texas Red (EX 560/55, EM 645/75). 2.7. 8-oxodG oligonucleotide pull-down assay 5 biotinylated 8-oxodG oligonucleotide, a 37mer containing a single 8-oxodG PF-06380101 residue at position 21 and control oligonucleotide having the same sequence as the 8-oxodG oligonucleotide except for the unmodified guanine at position 21, were custom synthesized by Sigma Genosys. Both oligonucleotides were subjected to duplex synthesis in individual reactions by incubating with equal amount of complementary strand oligonucleotide in annealing buffer (10 mM Tris [pH 7.6], 10 mM MgCl2, 1 mM EDTA) for 10 min at 75 C. Duplexes were then allowed to cool down at space temp and DNA concentration measured. The duplexes (750 ng) were then immobilized onto anti-biotin antibody labeled agarose beads by incubating with 20 l of the beads in binding buffer (20 mM Hepes [pH 7.9], 100 mM KCl, 2 mM MgCl2, 0.5 mM EDTA, 1 mM DTT, 0.4 mM ZnSO4, 40 M ZnCl2, 10% Glycerol and 0.1% Triton X-100) for 1 h at 4.