Journal: PLoS ONE

Article Title: Mitochondrial Localization of ABC Transporter ABCG2 and Its Function in 5-Aminolevulinic Acid-Mediated Protoporphyrin IX Accumulation

doi: 10.1371/journal.pone.0050082

Figure Lengend Snippet: Human lung adenocarcinoma cells (A549), human renal cancer cells (Caki-1),human colorectal cancer cells (DLD-1),human bladder cancer cells (T24) and human histiocytic lymphoma cells (U937) were used in this experiment. A, Western blotting analysis of whole cell lysates of various cancer cell lines. ABCG2 was detected with a polyclonal anti-ABCG2 (Cat. #4477; Cell Signaling Technology). Actin was used as a protein loading control. A representative blot of multiple experiments is shown. B , Relative contents of each protein were determined by densitometric scanning and were normalized using actin. C , Levels of accumulated ALA-mediated PpIX in various cancer cell lines. Bar graphs show flow cytometric analysis results of cellular PpIX content. Mean intensities of fluorescence (autofluorescence values) obtained from control experiments of each cell line were normalized to 100%. Results represent the mean ± SD of three independent experiments.

Article Snippet: Polyclonal antibodies against ABCG2 (Cat. #4477), cytochrome c oxidase subunit IV (CoxIV), Na, K-ATPase, GM130, and Alexa Fluor 488-conjugated goat anti-mouse IgG were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Fluorescence

Journal: PLoS ONE

Article Title: Mitochondrial Localization of ABC Transporter ABCG2 and Its Function in 5-Aminolevulinic Acid-Mediated Protoporphyrin IX Accumulation

doi: 10.1371/journal.pone.0050082

Figure Lengend Snippet: Live cells cultured on glass coverslips were incubated with 25 nM of Mito Tracker Red CMXRos to label mitochondria ( red ); then they were fixed and processed for ABCG2 protein immunostaining ( green ). Confocal images were obtained from cells immunostained with mouse monoclonal anti-ABCG2 antibody (5D3; R&D Systems) and Alexa Fluor 488-conjugated goat anti-mouse IgG antibody. Insets show a magnified area, indicated by white arrows.

Article Snippet: Polyclonal antibodies against ABCG2 (Cat. #4477), cytochrome c oxidase subunit IV (CoxIV), Na, K-ATPase, GM130, and Alexa Fluor 488-conjugated goat anti-mouse IgG were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Cell Culture, Incubation, Immunostaining

Journal: PLoS ONE

Article Title: Mitochondrial Localization of ABC Transporter ABCG2 and Its Function in 5-Aminolevulinic Acid-Mediated Protoporphyrin IX Accumulation

doi: 10.1371/journal.pone.0050082

Figure Lengend Snippet: A , Subcellular fractions were isolated by a centrifugation method as described in . ABCG2 was detected by western blotting analysis using polyclonal anti-ABCG2 (Cat. #4477; Cell Signaling Technology). CoxIV, Actin, SEC23A, GM130, and Na, K-ATPase were used as organelle-specific markers of mitochondria, cytosol, endoplasmic reticulum, Golgi apparatus, and plasma membrane, respectively. A representative blot of multiple experiments is shown. B , Highly purified mitochondria were isolated by OptiPrep density gradient centrifugation as described in and the western blotting analysis was performed using antibodies as described in A. Equal volume of samples from both whole cell lysate and mitochondrial fraction of A549 cells were subjected to SDS-PAGE. A representative blot of multiple experiments is shown.

Article Snippet: Polyclonal antibodies against ABCG2 (Cat. #4477), cytochrome c oxidase subunit IV (CoxIV), Na, K-ATPase, GM130, and Alexa Fluor 488-conjugated goat anti-mouse IgG were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Isolation, Centrifugation, Western Blot, Purification, Gradient Centrifugation, SDS Page

Journal: PLoS ONE

Article Title: Mitochondrial Localization of ABC Transporter ABCG2 and Its Function in 5-Aminolevulinic Acid-Mediated Protoporphyrin IX Accumulation

doi: 10.1371/journal.pone.0050082

Figure Lengend Snippet: A, Confocal immunofluorescence analysis of HEK cells and ST-HEK cells. Mitochondria, stained with Mito Tracker Red CMXRos; ABCG2, stained with monoclonal anti-FLAG IgG and Alexa Fluor 488-conjugated goat anti-mouse IgG. B, Western blotting analysis of mitochondrial fraction of HEK cells and ST-HEK cells. A representative blot of multiple experiments is shown.

Article Snippet: Polyclonal antibodies against ABCG2 (Cat. #4477), cytochrome c oxidase subunit IV (CoxIV), Na, K-ATPase, GM130, and Alexa Fluor 488-conjugated goat anti-mouse IgG were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Immunofluorescence, Staining, Western Blot

Journal: PLoS ONE

Article Title: Mitochondrial Localization of ABC Transporter ABCG2 and Its Function in 5-Aminolevulinic Acid-Mediated Protoporphyrin IX Accumulation

doi: 10.1371/journal.pone.0050082

Figure Lengend Snippet: Mitochondrial preparations of A549 cells (A) and ST-HEK cells (B) were treated with proteinase K on ice for 60 min in the presence or absence of 0.2% Triton X-100 (TX-100). Then the proteins were analyzed by Western blotting using a polyclonal anti-ABCG2 antibody. A representative blot of three independent experiments is shown.

Article Snippet: Polyclonal antibodies against ABCG2 (Cat. #4477), cytochrome c oxidase subunit IV (CoxIV), Na, K-ATPase, GM130, and Alexa Fluor 488-conjugated goat anti-mouse IgG were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Mitochondrial Localization of ABC Transporter ABCG2 and Its Function in 5-Aminolevulinic Acid-Mediated Protoporphyrin IX Accumulation

doi: 10.1371/journal.pone.0050082

Figure Lengend Snippet: Mitochondrial preparations of A549 cells (A) and ST-HEK cells (B) were treated with PNGase F or Endo H, and then ABCG2 was detected by Western blotting using a polyclonal anti-ABCG2 antibody. A representative blot of three independent experiments is shown.

Article Snippet: Polyclonal antibodies against ABCG2 (Cat. #4477), cytochrome c oxidase subunit IV (CoxIV), Na, K-ATPase, GM130, and Alexa Fluor 488-conjugated goat anti-mouse IgG were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot