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. 2011 Nov 16;6(11):e27465. doi: 10.1371/journal.pone.0027465

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Noor et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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Family of 14-3-3 proteins identified from silver stained gels (SS) and western blots (WB) of Fractions 1, 2 and 3 (F1, F2 and F3) and western blots of un-fractionated homogenate (WB) H of spinal cord segment caudal to the site of injury. Two subtypes, 14-3-3 epsilon and gamma were identified in silver stained gels and detected by mass spectrometry in F1, F2 and F3, pH 3.00–3.58, pH 3.58–4.16 and pH 4.16–4.76, respectively. 14-3-3 epsilon subtype was identified as the higher band (grey arrow) whilst 14-3-3 gamma subtype was identified as the lower band (black arrow). Results showed that 14-3-3 epsilon increased 7 days after injury at both ages (P7+7d and P28+7d) compared to age-matched controls (P14 and P35) in F1, while in F2 it increased 1 day after injury at P7 only (P7+1d). 14-3-3 gamma (lower band) was predominantly expressed in F2 at all ages but was additionally detectable in F1 at P35 and F3 at P8 and P29. In the P7-injured group, 14-3-3 gamma was initially down-regulated at 1 day after injury but was up-regulated 7 days after in F2. However in F3, it was up-regulated 1 day after injury but was not detectable at P14. In P28-injured group it showed differential expression in all fractions where it was down-regulated in F2 but was up-regulated in F3 one day after spinal injury. Seven days after injury, it was down-regulated in F1 but up-regulated in F2. Silver stained gels of F1-F3 were confirmed using pan-14-3-3 antibody and western blotting (WB). Western blotting of un-fractionated homogenates (H), which contain all 14-3-3 subtypes, with the pan-14-3-3 antibody showed no detectable overall changes in expression levels in the samples from spinal cords after injury compared to age-matched controls. Western blotting of un-fractionated homogenates with anti-14-3-3 epsilon showed that there was an increase in 14-3-3 epsilon expression at P7+1d and P7+7d compared to age-matched control. No changes were observed in P28-injured group compared to age-matched control. MW: Molecular weight marker (MW×10³).