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. 2016 Jun 7;7(29):45214–45224. doi: 10.18632/oncotarget.9901

Figure 5. A cyclin D1/ROS/ERK1/2 axis regulates cell adhesion.

Figure 5

(A) Whole-cell protein extracts were obtained from cultured GFP and D1-GFP clones and separated by SDS-PAGE. The proteins were blotted and analyzed using the indicated Abs. An anti-β-actin Ab was used as a loading control. Densitometric analyses were performed on the images captured with the ChemiDoc™ XRS+ molecular imager and analyzed using Image Lab™ software (Bio-Rad). The respective p-ERK1/2/ERK1/2 ratios of GFP- and D1-GFP-expressing clones are indicated under their respective lanes. (B) TAT-cyclin D1 fusion protein was produced in bacteria, purified, and directly added to the Ramos cell culture medium (or 0.9% NaCl as a control) as previously described [15]. The cells were harvested 3 or 24 h later for western blot analysis using the indicated Abs. Anti-β-tubulin Ab was used as a loading control. (C) D1-GFP-expressing clones (Cl2 and Cl4) were treated with 1 mM NAC for 24 h (or vehicle as a control) and harvested for protein purification and analysis after SDS-PAGE and immunoblotting as before with the indicated Abs. An anti-β-actin Ab was used as a loading control. (D, E) D1-GFP-expressing Cl2 and Cl4 were treated with 5 or 15 nM PD0325901 for 24 h, then assayed for cell adhesion on HS-5 stromal cells (D) and chemotaxism (E) as already described.