Validation of proteins identified by mass spectrometry.

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Validation of proteins identified by mass spectrometry. Validation of proteins identified by mass spectrometry. A subset of proteins identified by the mass spectrometry screen to be C/NM bound in G0 or in cells stimulated with PMA/ionomycin were analyzed by western blotting. The samples analyzed were (A, C, D), total cell lysates of T cells during the G0 or G1 transition (0–40 h +PMA/ionomycin); chromatin/nuclear matrix‐bound (Chr) and unbound (free) proteins in G0 or post‐stimulation (0 and 40 h +PMA/ionomycin); (B), chromatin/nuclear matrix‐bound (Chr) and free samples taken at each of the time points indicated. The patterns of expression and binding for proteins as indicated in (A) are as follows: (a) proteins, such as Topo I, RUNX3 and Mcm7 are present at a low/undetectable level in G0 and are induced and bind C/NM post‐stimulation; (b) proteins such as STAT3 and NF‐κB p50 are present at similar levels in G0 after stimulation but only bind chromatin in stimulated cells; (c) proteins such as Prohibitin and PARP‐1 are present and bind C/NM in G0 but the levels of binding increase when cells are stimulated with PMA/ionomycin. LSP‐1 is an example of a protein C/NM bound in G0 but is reduced in stimulated cells. Controls for cell‐cycle progression and C/NM‐bound versus ‐unbound proteins and sample loading (Histones stained with Coomassie Blue and p42/p44 MAPK (ERK1/2)) are shown. Representative of n>3 independent experiments for each protein. For each protein analyzed by western blotting, the fold‐change of the C/NM binding between cells in G0 and 40 h post‐stimulation with PMA/ionomycin, determined by mass spectrometry, is shown in Supplementary Table S8A. Source data is available for this figure in the Supplementary Information. Stephen J Orr et al. Mol Syst Biol 2012;8:573 © as stated in the article, figure or figure legend