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Expression proteomics of UPF1 knockdown in HeLa cells reveals autoregulation of hnRNP A2/B1 mediated by alternative splicing resulting in nonsense-mediated mRNA decay

Nicholas J McGlincy13, Lit-Yeen Tan1, Nicodeme Paul2, Mihaela Zavolan2, Kathryn S Lilley1 and Christopher WJ Smith1*

Author Affiliations

1 Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW, UK

2 Biozentrum, University of Basel, Klingelbergstr. 50-70, CH 4056 Basel, Switzerland

3 MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK

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BMC Genomics 2010, 11:565  doi:10.1186/1471-2164-11-565

Published: 14 October 2010

Additional files

Additional file 1:

Protein spots of interest excised for identification by mass spectrometry. Cy2 image of one gel from the 2D-DiGE multi-gel study. The 85 up-regulated spots excised for identification by mass spectrometry are circled in red and the 17 down-regulated spots excised are circled in blue. Each spot is labelled with its number from Additional file 2.

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Additional file 2:

XLS file containing the collated data from the 2D-DiGE multi-gel study, peptide sequence data and protein IDs produced by mass spectrometry, computational prediction of NMD activating features and QPCR based validation of changes in mRNA expression.

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Additional file 3:

A. Novel 3' UTR sequence of HNRNPA2B1 constructed from RACE tags. Exon sequence is capitalised. HNRNPA1 and A2/B1 motifs from [91] are emboldened and underlined. The small UTR intron present in the Refseq UTR is outlined in black. B. Sequences of the 3' RACE tags illustrated in Figure 6.

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Additional file 4:

List of all the QPCR primer used in this study, including sequence and design source.

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Additional file 5:

Sorting of GFP positive cells by flow cytometry. Representative plots of the cell sorting used in Figure 6. Cell events are denoted by dots. Increasingly "hot" colours represent increasing density of cell events. In each panel, the cells selected for further sorting are outlined by a polygon (termed a gate), and the inlaid number indicates the percentage of cells at that stage within the gate. A. Cells were gated based on forward and side scatter to eliminate debris. B. Doublet discrimination was carried out to ensure only single cells were sorted. C. Live/dead cells were discriminated with To-Pro-3 staining, detected using a 670/30 filter. D. GFP fluorescence was detected using a 530/30 filter.

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