supplemented with 10% fetal calf serum, 5 mM hypoxanthine, 20 M 4aminopteroic acid, and 0.8 mM thymidine. The human cervical cancer cell line HeLa was maintained in RPMI medium 1640 supplemented with 10% FCS. The hTERT1-immortalized human RPE cell line was grown in D-MEM / F12 medium containing 2.5 mM Lglutamine, 10% FCS, 0.25% sodium 15557325 bicarbonate. The human osteosarcoma cell line U2-OS ) was grown in D-MEM medium supplemented with 2% L-glutamine and 10% FCS. All cell lines were grown at 37 C in a humified atmosphere of 5% CO2. Mycoplasma contamination was ruled out by routine control testing using a luminescence-based detection kit. 2 miR-525-3p Mediated Survival after Irradiation Identification of deregulated proteins was done by the computer program Decyder as previously described. Protein spots were considered to be differentially regulated if a statistically significant difference in intensity was achieved at the 95% confidence level, and if the standardized average spot volume ratio exceeded 1.3-fold and p 0.05. Destaining of the silver stained 2D spots and in-gel trypsin digestion was performed prior to mass spectroscopy, as described previously. Mass spectra of abundant protein spots were acquired using MALDI-TOF/TOF mass spectrometry. For less abundant spots the identification was made by LC-MS/MS linear quadrupole ion trap-Orbitrap mass spectrometry. The configurations and the experimental set up of both machines were as described previously. The GPS Explorer TM Software was used for MALDI-TOF/TOF spectra analyses. Scaffold was used to validate MS/MSbased peptide and protein identifications obtained by LCMS/MS. Carbamidomethylation was set as the fixed purchase Scopoletin modification and oxidized methionine as the variable modification. The acquired MS/MS spectra were searched with Protein pilot software 3.0 against the Swiss-Prot database using Mascot 2.3.02 with the following parameters: As taxon we chose human and as enzyme trypsin allowing up to one missed cleavage. Peptide identifications were accepted if they were established with a greater than 80 % probability as specified by the Peptide Prophet algorithm. Proteins were identified if they showed 11693460 a greater than 95.0% probability and contained at least 2 unique identified peptides. In silico identification of potential miR-525-3p target sequences The FUZZNUC program was used to search for complete complementarities to the 8mer, 7mer-A1 and 7mer-m8 seed sequences of miR-525-3p. Putative target genes were predicted using five different software tools, namely TargetScan, RNA22, MicroCosm, miRWalk and DIANA-microT. Luciferase reporter assay to identify mRNAs directly targeted by miR-525-3p Candidate gene cDNA sequences were obtained by PCR amplification of reverse transcribed EA.hy926 cell mRNA using the primer sets indicated in Immunoblot analysis Immunoblotting was performed for the validation of deregulation of selected proteins. EA.hy926 cells were lysed in RIPA buffer supplemented with 1 mM protease inhibitors for 20 min on ice. Western blot analysis was accomplished according to standard procedures using enhanced chemiluminescence detection. For detection of the proteins primary antibodies directed against beta-arrestin-1, thioredoxin 1, heterogeneous nuclear ribonucleoprotein K and the 70kDa heat shock protein 9 were used. Protein loading was monitored by the detection of actin or PCNA. HRP conjugated anti-mouse, anti-goat or anti-rabbit antibodies were used to reveal binding of primary

Datasets were exported into Microsoft Excel for further calculations

About author

Leave a reply

You must be logged in to post a comment.