The data presented here shows

The data presented here shows that inhibition of the 26S proteasome causes significant changes in the expression of Card10, Dffb, Traf3 and Trp53bp2 genes. In addition, four genes were found consistently downregulated in response to treatment with bortezomib for 24h. The genes downregulated were Bcl2-like 1, Fas (TNFRSF6)-associated via death domain, Tnf receptor-associated factor 1 and X-linked inhibitor of apoptosis proteins (Table 1). Western blotting – The ECL Western blotting kit was used according to manufacturer procedure (GE Healthcare, Stockholm, Sweden). A total of 50μg protein from each sample was separated on a 12% SDS-PAGE. Afterwards, proteins were transferred to PVDF membranes at 70V for 2h. After the transfer, the PVDF membranes were washed briefly with methanol and left for drying for 15min to enhance the protein binding. The PVDF membranes were again reactivated by methanol. The membranes were blocked by 5% non-fat dried milk in TBS-T. The membranes were then incubated with anti-Card10 (1:500) and anti-Trp53BP2 (1:500) for 1h. For loading control, the membranes were probed with anti-β-actin antibody (1:5000) in TBS-T for 1h. The membranes were then incubated with HRP-conjugated anti-rabbit secondary antibody (1:5000 dilution) in TBS-T for 1h. The Western blot analysis results indicated that the increases in Card10 (Fig. 1A, upper panel) and Trp53bp2 (Fig. 1A, middle panel) proteins were corroborated in response to proteasomal inhibition by various concentrations of bortezomib for 24h. The examination of β-actin level (Fig. 1A, lower panel) showed that the changes in the protein levels of Card10 and Trp53bp2 were not simply due to higher protein loading. As can be seen in Fig. 1B, when the SLx-2119 were treated with different doses of bortezomib, a threshold-dependent increase in Card10 protein was clearly observed. With 10nM, 50nM, 100nM and 200nM, 1.84 fold, 2.31 fold, 2.26 fold and 3.78 fold increases were detected, respectively. On the other hand, the increase in the level of Trp53bp2 protein was observed with only higher doses of bortezomib (i.e., 100nM and 200nM) (Fig. 1B).
Specifications table
Collection of uterine fluid samples and preparation for MS analyses Uterine fluid were collected as described previously [3] on brown-egg laying hens at 7h, 14h and 22h after previous oviposition (p.o). These time intervals correspond to the initiation (I), rapid growth (G) and termination (T) phases of shell mineralization, respectively. For global protein inventory, equal amounts of protein from uterine fluids collected at each stage of shell calcification were pooled. A total of 135µg of proteins was fractionated on a 4–20% SDS-PAGE gel (8.3cm×7.3cm×1.5mm). Proteins on gels were stained with Coomassie blue and the entire SDS-PAGE lanes were sectioned into 20 bands. For quantitative analyses, 18 individual uterine fluid samples were used. Six uterine fluids collected at the same stage were pooled in equal amount of proteins for each stage (initial (I), growth (G) and terminal (T) stage samples). The three samples I, G and T (112µg of proteins/sample) were applied to a 4–20% SDS-PAGE gel (8.3cm×7.3cm×1.5mm). A brief migration without fractionation was performed until samples were concentrated in a narrow band. The three resulting protein bands were stained with Coomassie blue and excised. Excised proteins for both approaches were in-gel digested with bovine trypsin (Roche Diagnostics GmbH, Mannheim, Germany), as previously described [4], and analyzed by nanoscale liquid chromatography-tandem mass spectrometry (nano LC–MS/MS).
Nano LC MS/MS analyses All experiments were performed on a linear ion trap Fourier Transform Mass Spectrometer (FT-MS) LTQ Orbitrap Velos (Thermo Fisher Scientific, Bremen, Germany) coupled to an Ultimate® 3000 RSLC Ultra High Pressure Liquid Chromatographer (Dionex, Amsterdam, The Netherlands) as previously described [4]. Raw data files were converted to MGF as previously described [4]. The identification of proteins was established using MASCOT search engine (v 2.3, Matrix Science, London, UK). The peptide and fragment masses obtained were matched automatically against IPI chicken (version 3.81) and against the chordata section of nr NCBI database (1601319 sequences, downloaded on 2012/03/22) and UniprotKB SwissProt (535248 sequences, downloaded on March 2012). Enzyme specificity was set to trypsin with two missed cleavages using carbamidomethylcysteine, oxidation of methionine and N-terminal protein acetylation as variable modifications. The tolerance of the ions was set to 5ppm for parent and 0.8Da for fragment ion matches. Mascot results obtained from the target and decoy databases searches were incorporated in Scaffold 3 software (version 3.4, Proteome Software, Portland, USA). Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm [5]. Peptides were considered distinct if they differed in sequence. Protein identifications were accepted if they could be established at greater than 95.0% probability as specified by the Protein Prophet algorithm [6] and contained at least one sequence-unique peptide for global inventory and at least two sequence-unique peptides for quantitative analysis. A false discovery rate was calculated as <1% at the peptide or protein level. The abundance of identified proteins was estimated by calculating the emPAI using Scaffold 4 Q+ software (version 4.2, Proteome Software, Portland, USA). Additionally, to perform label-free quantitative proteomic analyses based on spectral counting method, Scaffold 3 Q+ software (version 3.4, Proteome Software, Portland, USA) was used to quantify the proteins at the three different stages of calcification. All proteins with greater than two sequence-unique peptides, identified in database with high confidence were considered for protein quantification. To eliminate quantitative ambiguity within protein groups, we ignored all the spectra matching any peptide which is shared between proteins. Thereby, quantification performed with normalized spectral counts was carried out on distinct proteins. Data have been deposited to the ProteomeXchange Consortium [2] via the PRIDE partner repository with the dataset identifier PXD000992.