Figure 3.

Inhibition of proliferative signaling increases PARK2 protein levels. A, Schematic presentation showing proliferative signaling pathway and inhibitors. Indicated cells were cultured with either C75 (B), Ly284002 (D), Rapamycin (E), MK2206 (F), or PF470851 (G) for 16 hours or infected with shRNA against FASN (C) and lysed. Posttreatment/infection, cell lysates were probed for PARK2 expression. GAPDH was used as the loading control. The bar diagram beneath each figure represents densitometric quantification of the immunoblots. Values were normalized with corresponding loading control and neutralized with corresponding DMSO (0.1%) or SCR-infected cells, which was set as 1; represented as mean ± SD for n = 3. Statistical analysis was performed using one-way ANOVA followed by Dunnett post hoc analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.005; ****, P < 0.001 versus corresponding cells. H and J, Toledo and Farage were treated with indicated PI3K signaling inhibitors [LY294002: PI3K inhibitor (1 μmol/L), AZD5363: Akt inhibitor (500 nmol/L), Rapamycin: mTOR inhibitor (50 nmol/L), Torin 1: mTOR inhibitor (250 nmol/L), PF-4708671: S6Kinase inhibitor (1 μmol/L)] in presence or absence of C75. Posttreatment, cell lysates were probed for PARK2. GAPDH was used as the loading control. I and K, Densitometric quantification of the immunoblots in H and J. Values were normalized with corresponding GAPDH and neutralized with DMSO-treated corresponding cells, which was set to 1. Values were represented as mean ± SD for n = 3. Statistical analysis was performed using Student t test (unpaired two-tailed) *, P < 0.05; **, P < 0.01, versus corresponding treated cells.