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. 2014 Mar 21;9(3):e92793. doi: 10.1371/journal.pone.0092793

Figure 4. Involvement of PTEN and PI3K in pRb2 hypophosphorylation induced by Ras/c-Raf inactivation.

Figure 4

(A) Cells were pretreated with bpv (an inhibitor of PTEN) for 30 min or YS-49 (an activator of PI3K) for 1 h, followed by 1 h of 100 nM 3MC treatment. Total cell lysates, and extraction of nuclear and cytosolic proteins were analyzed using Western blot analysis for related signaling molecules involved in chromatin deaceylation. (B) Cells were transfected with a plasmid containing the DNRas gene to mimic the effect of 3MC. After 1 h of 3MC treatment, cells were harvested for Western blot analysis of the mentioned molecules in total and cytosolic-membrane extracts. GAPDH, Lamin A/C, and VE-cadherin were used as internal controls for the cytosol (or total), nuclear and membrane fractions, respectively, to verify equivalent loading. The lower panel shows the intensity of bands in the Western blots using densitometry. Data are presented as mean ± SEM of 3 independent experiments (*P<0.05 vs. control group; # P<0.05 vs. 3MC treatment alone).