Figure 4.

The pattern and intensity of protein expression of both activated AKT (pAKTS473) and pFOXO3a(S253) follows the PML–PHLPP2 signaling axis, which is governed by the activity status of CK2. (a) (ii) Both knockdown of PML and PHLPP2 (i) maintains a higher nuclear level of pAKTS473. PC3 cells were transfected with either scrambled si RNA (Control) or PML si or PHLPP2 si. Cells were harvested and fractionated into CE and NE. The lysates were separated on SDS-PAGE, followed by IB with anti-pAKTS473 antibody. (b) AKT-S129D is enhanced in the nucleus under normal condition, while it is significantly depleted from NE fraction upon inhibition of CK2 activity. HEK293 cells were transfected with either WT-AKT or AKT-S129D vectors or the latter also being treated with TBCA. Cells were harvested and fractionated into CE and NE. The lysates were subjected to IB analysis with anti-pAKTS473 antibody. (c) The level of AKT-S129D is lowered in the nucleus by exogenously overexpressed PML. HEK293 cells were transfected with AKT-S129D. Same cells were either transfected with WT-CK2α or WT-PML or treated with TBCA. CE and NE fractions were prepared and subjected to IB analysis with anti-pAKTS473 antibody. Cells treated with TBCA served as positive control. (d) Both knockdown of PML and PHLPP2 results into heightened level of pFOXO3a. PC3 cells were transfected with either scrambled si RNA or PML si or PHLPP2 si. Lysates were prepared from cells, harvested after 72 h of post-transfection and subjected to IB analysis, being probed with the indicated antibodies. (e) Activation of CK2 similarly results into increase in the level of pFOXO3a, while inhibition of CK2 produces the antagonistic effect. PC3 cells were treated with either d—Sorbitol or TBCA. Lysates were obtained after indicated duration of time and analyzed through IB by probing with mentioned antibodies. (f) Exogenous overexpression of WT-CK2α and WT-PML has antagonistic effect on the level of pFOXO3a. PC3 cells were transfected with either WT-CK2α or WT-PML and lysates were prepared and separated on SDS-PAGE followed by IB with the indicated antibodies. (g) AKT-S129D increases FOXO3a phosphorylation at Ser253 residue, while reversal of the phenomenon occurs with simultaneous inhibition of CK2. PC3 cells were transfected with AKT-S129D and also simultaneously treated with either d—Sorbitol or TBCA. Lysates were immunoblotted with the indicated antibodies. Control cells (in b, c, e) were treated with empty vector along with appropriate amount of DMSO-water