Analysis of Akt and Wnt/β-catenin signaling in <i>Tcl1-</i>deficient and -overexpressing ES cells.

<p>(A) Western blot analysis of GSK, Akt, and β-catenin in wild-type (WT), <i>Tcl1^−/−</i> (KO) #2 and #4, <i>Tcl1^−/−</i>(CAG-<i>Tcl1</i>) #1 and #3, and <i>Tcl1^−/−</i>(CAG-EGFP) #5 ES cells. (B) Western blot analysis of active β-catenin, Oct3/4, and HSP90 in the cytoplasmic (C) and nuclear (N) fractions of wild-type (WT), <i>Tcl1^−/−</i> (KO) #2 and #4, and <i>Tcl1^−/−</i>(CAG-<i>Tcl1</i>) #10 and #1 ES cells. <i>Tcl1^−/−</i>(CAG-<i>Tcl1</i>) #10 and #1 were derived from <i>Tcl1^−/−</i> (KO) #2 and #4, respectively. Proper fractionation was confirmed by western blotting of Oct3/4 and HSP90, which localize to the nucleus and cytoplasm, respectively. Be8cause active β-catenin levels in the nuclear fractions were much lower than those in the cytoplasmic fractions, active β-catenin in the nuclear fractions was detected by approximately two-fold longer exposure compared with that in the cytoplasmic fractions. (C) TOPflash assay. <i>P</i> values of wild-type ES cells (WT) compared with <i>Tcl1^−/−</i>(CAG-<i>Tcl1</i>) #1 and #3 ES cells were less than 0.01. <i>P</i> values of <i>Tcl1^−/−</i> (KO) #4 and #5 ES cells compared with <i>Tcl1^−/−</i>(CAG-<i>Tcl1</i>) #1 and #3 ES cells were less than 0.02.