The instrument was operated in a data dependent mode automatically switching between MS, MS2, and pdMS3. The top 10 parent ions of the spectra were chosen for fragmentation. The pdMS3 Olaparib PARP inhibitor acquisition was set to automatically select and further fragment the frag ment ion originating from the loss of phosphoric acid from the parent ions. Database analysis The . raw MS data were processed using the ThermoProteome Discoverer software. The generated. mgf files were subsequently searched against the murine sequence li brary in the International Protein Index protein se quence database using an in house Mascot server. The search was performed by choosing trypsin as the enzyme with two miss cleavages allowed. Carbamidomethyl, dimethyl labeling for light, medium and heavy modi fications of N terminus and Lys were chosen as the fixed modification.
As variable modifications, oxidations and phosphoryl ation, were chosen. The data were searched with a peptide mass tolerance of 10 ppm and a fragment mass tolerance of 0. 8 Da. A concatenated decoy database search was performed in a concatenated decoy mouse database de rived from the IPI mouse database listed above for each of the conditions, and only peptides with up to 1% of False Discovery were selected. Dimethyl quantification was performed using Thermo Proteome Discoverer from the extracted chromatograms obtained. Normalization was achieved using the LOWESS fitting al gorithm and protein grouping and statistics were obtained using StatQuant.
The phosphopeptide subpopulation were compared to a databasis consisting of motifs for phosphorylation by different kinases in NetworKIN website Total mRNA extraction and purification from rhBMP2 induced msMSC cells 3. 104 cells per ml were seeded onto 100 mm diameter Carfilzomib cul ture plate. After treatment with rhBMP2 for different time periods, cells were washed with ice cold PBSA, and total mRNA was isolated using silica columns from the RNeasy mini kit. The mRNA concentration was determined by absorbance at 260 nm and the purity of the preparations was evaluated by the A260nm A280nm ratio, with purity being considered when this ratio was approximately 2. 0. cDNA synthesis Total cellular RNA, isolated as mentioned above, was used to synthetize the corresponding cDNA. An aliquot of RNA from each condition was incubated with 2 units of DNase I and 20 units of RNAseOUT for 10 min at 37 C. After this incubation period, both enzymes were heat inactivated for 10 min at 75 C and 1 ul of 0. 5 ug ul of oligo dT, 1 ul of 10 mM dNTP, were added. The samples were incubated for 10 min at 65 C and then immediately placed on ice.
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