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. 2015 Sep 28;5:14534. doi: 10.1038/srep14534

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Copyright © 2015, Macmillan Publishers Limited

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(A) The activation status of Rac was compared in wild-type and Tsc2^−/− MEFs by pull down of Rac-GTP using recombinantly-produced PAK-Crib proteins. Tsc2^−/− MEFs are not characterised by excessive Rac activity. (B) Rac-GTP pull down with recombinantly-produced PAK-Crib proteins was used to investigate the effect of a 30 min treatment of 20 μM NSC23766 on Rac activity. The treatment efficiently blocks GTP loading of Rac. (C) The effects of a 1 hour treatment with 20 nM rapamycin and a 30 min treatment with 20 μM NSC23766 on Pak2 autophosphorylation and S6 phosphorylation were investigated in wild-type and Tsc2^−/− MEFs. Rapamycin efficiently inhibits S6 phosphorylation but has no effect on Pak2 autophosphorylation. NSC23766 affects neither Pak2 autophosphorylation nor S6 phosphorylation. The combination of rapamycin and NSC23766 (lanes cropped from the same blot) does also not influence Pak2 phosphorylation, nor is it more effective on S6 phosphorylation as compared to rapamycin alone. All blots were run under the same conditions and analysed in a similar way.