Journal: BMC Cell Biology

Article Title: Evidence for a mitochondrial localization of the retinoblastoma protein

doi: 10.1186/1471-2121-10-50

Figure Lengend Snippet: Rb protein is localized in mitochondria . A . Equal protein amounts (40 μg) of total extract (T), nuclear (N) and mitochondrial (M) fractions of human (HT1080 and HF) and rat (PC12 and FR3T3) cells cultured either untreated or etoposide-treated (16 h), were loaded onto gel and then immunoblotted with: anti-Rb (G3-245) antibody, the mitochondrial (COX II or cytochrome c), the cytosolic (Tubulin) or the nuclear marker antibodies (PCNA or Lamin A). HyperPh or hypoPh represents the phosphorylated state of Rb. ( Lower panel ) The quantification (Image J software) of the Rb and the PCNA protein levels (or Lamin A for FR3T3) in nuclear and mitochondrial fractions illustrated as mitochondrial/nuclear ratio (untreated cells). B . Effect of Rb siRNA on the presence of Rb in the mitochondria. FR3T3 cells were incubated for 48 h with Rb siRNA (siRb), control siRNAs (siCtrl) or non-transfected (NT). The total cell extract and mitochondrial fractions (20 μg) were loaded onto gel and subjected to immunoblotting with anti-Rb (G3-245) antibody. Quantifications were performed with respect to Enolase (for the total extract) and COX II (for the mitochondrial extract). Student's tests were performed (** P < 0.01).

Article Snippet: Then, 10 nM Rb siRNA (Rn_Rb1_1_HP siRNA, Qiagen) or negative control (non-silencing, Qiagen) and HiPerFect Transfection Reagent (Qiagen) were mixed in culture medium and then added to the cells, according to the manufacturer's recommendations (HiPerFect Transfection Reagent Handbook, Qiagen).

Techniques: Cell Culture, Marker, Software, Incubation, Control, Transfection, Western Blot