All search results were loaded into Scaffold software (Version 3

All search results were loaded into Scaffold software (Version 3.3.1; Proteome Software, Portland, OR) for comparative analyses using spectral counting of tandem mass spectra and full annotation of the data (Searle, 2010). which RUNX2 forms functional complexes with BAZ1B, RUVBL2 and INTS3 to mount an integrated response to DNA damage. This proposed cytoprotective function for RUNX2 in malignancy cells might clarify its expression in chemotherapy-resistant and/or metastatic tumors. 400C2000 range, followed by serial tandem mass spectrometry (i.e. MS/MS) around the seven most abundant signals. Precursor ion isolation width was 2.0?Da, collision energy was 35%, ion populace targets were 10,000 for MS and 5000 for MS/MS, and maximum ion fill occasions were 200?ms for both acquisition types. Precursor ions analyzed were subjected to dynamic exclusion for 30?s using a windows of ?0.5 to +1.5 371 and 445 were also excluded from MS/MS. Another set of similarly prepared samples were analyzed using a Proxeon Easy nanoLC (Thermo Scientific) system directly configured to an LTQ-Orbitrap Velos (Thermo Scientific) hybrid mass spectrometer. Peptide samples (2?l) were loaded at 4?l/min for 7?min onto a custom-made trap column (100?m i.d. fused silica with Kasil frit) made up of 2?cm of 200-?, 5-m Magic C18AQ particles (Michrom Bioresources, Auburn, CA). Peptides were then eluted using a custom-made analytical column (75?m i.d. fused silica) with a gravity-pulled tip and packed with 25?cm of 100-?, 5-m Magic C18AQ particles (Michrom). Peptides were eluted with a linear gradient as explained above. Mass spectrometry data were acquired using a data-dependent acquisition routine of acquiring one mass spectrum from 350C2000 in the Orbitrap (resolution, 60,000; ion populace, 1.0106; maximum ion injection time, 500?ms) followed by tandem mass spectrometry in the linear ion trap (LTQ) of the ten most abundant precursor ions observed in the mass spectrum. MS/MS data were acquired using a precursor isolation width of 2.0?Da, a collision energy of 35%, an ion populace of 5000 and a maximum ion fill time of 50?ms. Charge-state rejection of singly charged ions and dynamic exclusion was utilized [?0.1 to +1.1?Da windows, repeat count 1 (30-s delay)] to minimize data redundancy and maximize peptide identification. The natural data files were processed using Extract MSN software (Thermo Scientific) and searched against the human index of the SwissProt database (version 09/24/11) with Mascot (version 2.3.02; Matrix Science, London, UK) and X! Tandem [The GPM (www.thegpm.org); version Cyclone (2010.12.01.1)] software packages. LTQ Orbitrap Velos data were searched using a parent mass tolerance of 15?ppm and a fragment mass tolerance of 0.5?Da. LTQ data utilized a parent tolerance of 1 1.2?Da and a fragment tolerance of 1 1.0?Da. Full tryptic specificity with two missed cleavages was considered, and variable modifications of acetylation (protein N-term), cyclization of N-terminal S-carbamoylmethylcysteine (peptide N-term) and oxidation (methionine) and fixed modification of carbamidomethylation (cysteine) were considered. All search results were loaded into Scaffold software (Version 3.3.1; Proteome Software, Portland, OR) for comparative analyses using spectral counting of tandem mass spectra and full annotation of the data (Searle, 2010). Peptide identifications were accepted if they could be established at 95% probability by the Peptide Prophet algorithm (Keller et al., 2002) following Scaffold delta-mass correction. Protein identifications were accepted if they could be established at 99% probability and contained at least two recognized peptides; protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii et al., 2003). Normalized spectral counts were calculated by dividing the spectral counts for an recognized protein by the sum of the spectral counts per sample. Quantitative gene expression analysis RNA was isolated using Trizol reagent (Thermo Fisher Scientific) and trace DNA was removed using the DNA-free RNA kit (Zymo Research, Irvine, CA). cDNA was synthesized using Superscript III (Thermo Fisher Scientific) and amplified using gene-specific primers (supplementary material Table?S2) and iTAQ SYBR green supermix (Bio-Rad Laboratories). Reactions were run and data collected Cytochrome c – pigeon (88-104) on an ABI PRISM 700 system (Thermo Fisher Scientific). Primers for PCR are displayed in supplementary material Table?S1. Immunoprecipitation Confluent cells on 100-mm plates were harvested and solubilized in lysis buffer (10?mM Tris-HCl pH?8.0, 100?mM NaCl, 1?mM EDTA, Rabbit Polyclonal to GPRIN2 0.1% Nonidet P40, 1?mM DTT and 1?mM PMSF). Insoluble material was removed by centrifugation. The supernatants were incubated for 12?h at 4C with 2?g of anti-RUNX2 (M70 or S19; Cytochrome c – pigeon (88-104) Santa Cruz Biotechnology, Santa Cruz, CA), anti-FLAG (M2; Sigma-Aldrich) and a mixture of anti-rabbit- and anti-goat-IgG (Santa Cruz Biotechnology). Following the addition of 30?l of Protein-A/GCagarose beads (Santa Cytochrome c – pigeon (88-104) Cruz Biotechnology), mixtures were incubated for 2?h at 4C with rotation. Immune complexes were washed three times with lysis buffer; the agarose beads were then boiled for 10?min in sample buffer. Immunoprecipitates were run on 8% acrylamide SDS-PAGE or 4C20% gradient precast gels (Bio-Rad), followed by Western blot analysis with antibodies against.