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. 2016 Feb 3;36(5):1502–1515. doi: 10.1523/JNEUROSCI.1930-15.2016

Figure 1.

Figure 1.

Changes in CN and NFAT signaling properties at 7 d after CCI. A, Representative Western blot for the CN A subunit in rat hippocampal whole-cell lysates from rat contralateral and ipsilateral hemispheres at 7 d after CCI. Whole-cell lysates from primary astrocyte cultures infected with adenovirus (Ad) expressing a constitutively active 45 kDa CN fragment (aCN) were run in parallel. Injury was associated with an increase in the proteolysis of CN to a 45–48 kDa fragment, similar in size to the fragment found in Ad-aCN-infected astrocyte cultures. B, Mean ± SD level (percentage contralateral hemiphere levels) of proteolysis in hippocampal homogenates in contralateral and ipsilateral hemispheres. p < 0.05, paired t test. C, Representative EMSA for hippocampal homogenates from the ipsilateral and contralateral hemispheres of rats exposed to CCI or to sham surgery. NFAT-binding probe was added to homogenates with and without antibodies (Ab) to each of the 4 CN-dependent NFAT isoforms to show supershifts and/or block shifts. Unlabeled WT (wt) and mutant (mt) DNA probe was included in some conditions to demonstrate DNA-binding specificity of the labeled probe. Bands denoted by the blue arrowheads reflect total NFAT binding. Red asterisks point to bands that were supershifted in response to inclusion of the NFAT1 antibody. Blue asterisks appear next to lanes where the NFAT4 antibody was included with the DNA-binding probe and illustrate a clear block shift. The black arrowheads point to bands that were relatively insensitive to antibody treatment conditions and likely reflect nonspecific binding. No gel shifts or block shifts were seen after inclusion of the NFAT 2 and 3 antibodies, suggesting that these isoforms did not significantly contribute to total NFAT binding under these conditions. D, Mean ± SD DNA-binding activity expressed as percentage of sham contralateral condition levels. NFAT1 was assessed from the supershifted bands obtained with the NFAT1 antibody (see red asterisks in C). NFAT 4 was assessed from the bands associated with blue arrowheads in C. n.s., Nonsignificant ipsilateral versus contralateral; *p < 0.05; + p < 0.01 ipsilateral versus contralateral, Fisher's PLSD test.