Figure 2

Rb localization at mitochondrial level. Rb localization at mitochondrial level: Mitochondria (MT) were isolated from PC12 cells and then subjected to subfractionation into mitoplast (MP) (the inner-membrane and matrix), inter-membrane space (IS) and outer-membrane (OM) using a conventional subfractionation protocol. The total extract (T), the nuclear fraction (N) and the cytosolic fraction (C) were loaded onto gel in parallel. Equal amounts (40 μg) from each fraction were loaded onto gel and submitted to immunoblot analysis using anti-Rb (G3-245) antibody to detect endogenous Rb. Fraction enrichment was tested using antibodies against the β-subunit of F1-ATPase and ANT for mitoplasts, and against VDAC and uMtCK for the outer-membrane. The cytosolic and nuclear contamination was assessed using anti-Actin and anti-TFIID antibodies. HyperPh or hypoPh represents the phosphorylated state of Rb. This data is representative for 2 independent experiments.