Journal: Nucleic Acids Research

Article Title: PARG is dispensable for recovery from transient replicative stress but required to prevent detrimental accumulation of poly(ADP-ribose) upon prolonged replicative stress

doi: 10.1093/nar/gku505

Figure Lengend Snippet: RPA binding to DNA is directly affected by PAR. ( A ) Anti-PAR antibodies co-immunoprecipitated RPA2 and RPA1 subunits of RPA at higher levels from HU-treated shPARG cells compared to shCTL cells. Cell extracts from shCTL (C) and shPARG (P) cells treated with 2 mM HU for 24 h were immunoprecipitated with anti-PAR antibodies. The presence of PARP-1, RPA1 and RPA2 in the anti-PAR immunoprecipitates (IP PAR, lanes 3 and 4), in the control immunoprecipitates using rabbit anti-mouse antibody (IP IgG, lanes 1 and 2) and in the input (lanes 5 and 6, corresponding to 5% of the lysate) was assessed by western blotting using the indicated antibodies. The asterisk points to a non-specific band. ( B ) PARP-1 PARylated by RPA mostly on RPA1 subunit. The trimeric RPA (2.5 μg, lanes 2, 3, 5 and 6) was incubated with 0 (lanes 2 and 5) or 500 ng of PARP-1 (lanes 3 and 6) for 30 min at 25°C in the presence of 32 P-NAD + and DNAse I-activated calf thymus DNA. As negative control, PARP-1 was incubated with GST (2 μg) (lanes 4 and 7). Reaction products were analysed by 8–20% SDS-PAGE and autoradiography (right panel) of the Coomassie blue-stained and dried gel (left panel). Lane 1: molecular weight marker. ( C ) RPA binds non-covalently to PAR. RPA complex (2.5 μg, lanes 2 and 6), GST (2 μg, lanes 3 and 7), H2A and H3 (1 μg each, lanes 4 and 8) and H2B, H4 (1 μg each, lane 5 and 9) were separated on 8–20% SDS–PAGE and either stained with Coomassie blue (left panel) or blotted onto nitrocellulose membrane, renaturated and incubated with free PAR. Bound PAR was detected by immunodetection using anti-PAR antibody (right panel, PAR-blot). Lane 1: molecular weight marker. ( D ) PARylated proteins are co-immunoprecipitated with RPA2 in HU-treated shPARG cells. Cell extracts from shCTL (C) and shPARG (P) cells treated with 2 mM HU for 24 h were immunoprecipitated with anti-RPA2 antibodies as described in “ Material and Methods ” section. The presence of PARylated proteins, RPA1 and RPA2 in the anti-RPA2 immunoprecipitates (IP RPA2, lanes 3 and 4), the control immunoprecipitates using mouse anti-HA antibody (IP IgG, lanes 1 and 2) and the input (lanes 5 and 6, corresponding to 5% of the lysates) was assessed by western blotting using the indicated antibodies. The asterisk points to non-specific bands. ( E and F ) EMSA analyses showing that RPA binding to ssDNA is favored by low levels but counteracted by high levels of PAR. ( E ) PAR competes with DNA for RPA. RPA (1.7 nM) was incubated with 1 nM of a Cy5.5-oligodT probe (50-mer) and increasing concentrations of PAR (lanes 3–11: from 8 ng in lane 3, doubled each point to 256 ng in lane 12) for 30 min at RT, complexes were separated by 1% native agarose gel electrophoresis and observed at 680 nm on Odyssey Infrared Imaging System. Lane 1: DNA only; lane 2: no PAR. F . High PAR levels dislodge RPA from DNA. RPA (7 nM) was pre-incubated with 2.5 nM of a Cy5.5-oligodT probe for 15 min before the addition of increasing concentrations of PAR (from lane 3 to 11: 4, 8, 16, 32, 48, 64, 96, 128, 192 ng, respectively) and incubation for 30 min at 23°C. Complexes were separated by 1% native agarose gel electrophoresis and observed at 680 nm on Odyssey Infrared Imaging System. Lane 1: DNA only; lane 2: no PAR.

Article Snippet: After washings, membranes were incubated for 1 h with the appropriate secondary antibodies: an Alexa Fluor 680 goat anti-rabbit or goat anti-mouse Alexa Fluor 680 (1:20 000, Invitrogen) or a Donkey anti-mouse IRDye800 (1:10 000, Li-Cor, Bioscience) and revealed on Odyssey Infrared Imaging System (Li-Cor, Bioscience).

Techniques: Binding Assay, Immunoprecipitation, Western Blot, Incubation, Negative Control, SDS Page, Autoradiography, Staining, Molecular Weight, Marker, Immunodetection, Agarose Gel Electrophoresis, Imaging

Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: (A) Cell homogenates ( H ) from the indicated cell lines were used to prepare cytosolic ( C ) and membrane ( M ) fractions. Equivalent amounts of each fraction (10 μg of proteins) were subjected to SDS-PAGE and immunoblotting using antibodies to the proteins indicated on the right. The position of molecular mass markers is indicated on the left. (B) Densitometric quantification of the immunoblot signal of the levels of GOLPH3 in the cell homogenates as shown in (A). (C) Densitometric quantification of the immunoblot signal of the levels of GOLPH3 in cytosolic ( C ) and membrane ( M ) fractions as shown in (A). Bar represents the mean ± standard deviation of the amount of immunoblot signal normalized with the signal for β-actin, and also for the total amount of protein in each fraction (for more details see ). *** P < 0.001; ns , not statistically significant.

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Membrane, SDS Page, Western Blot, Standard Deviation

Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: (A-C) Samples of a membrane fraction (70 μg of proteins) from MCF 10A (A), MCF7 (B), and MDA-MB-231 (C) cells were incubated on ice for 1 hour with either 10 mM Tris HCl pH 7.4 ( Control ), 1 M KCl in 10 mM Tris HCl pH 7.4 ( KCl ) or 0.2 M Na 2 CO 3 pH 11.3 ( Na 2 CO 3 ). After centrifugation, pelleted membranes ( P ) and extracted proteins in the supernatant ( S ) were processed by SDS-PAGE and immunoblotting using antibodies to the proteins indicated on the right. Syn16 , Syntaxin 16. The position of molecular mass markers is indicated on the left. (D-F) Densitometric quantification of the immunoblot signal of the levels of GOLPH3 in pellets ( P ) and supernatants ( S ) as shown in A-C of membranes incubated in control conditions (D), in 1 M KCl (E), or in 0.2 M Na 2 CO 3 (F). Bar represents the mean ± standard deviation of the amount of immunoblot signal. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Membrane, Incubation, Control, Centrifugation, SDS Page, Western Blot, Standard Deviation

Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: NRK (A and B), MCF 10A (C and D), MDA-MB-231 (E and F), and MCF7 (G and H) cells were left untreated ( Control ) or treated with 5 μg/ml BFA for 60 min ( BFA ). Cells were fixed, permeabilized, and immunolabeled with rabbit polyclonal antibody to GOLPH3, mouse monoclonal antibody to GM130, and either sheep antibody to TGN38 (A and B) or sheep antibody to TGN46 (C to H). Secondary antibodies were Alexa-594-conjugated donkey anti-rabbit IgG (red channel), Alexa-488-conjugated donkey anti-mouse IgG (green channel), and Alexa-647-conjugated donkey anti-sheep IgG (blue channel). Stained cells were examined by fluorescence microscopy. Merging red, green, and blue channels generated the fourth image on each row; yellow indicates overlapping localization of the red and green channels, cyan indicates overlapping localization of the green and blue channels, magenta indicates overlapping localization of the red and blue channels, and white indicates overlapping localization of all three channels. Insets show 1.7x magnifications. Bar, 10 μm.

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Control, Immunolabeling, Staining, Fluorescence, Microscopy, Generated

Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: (A-C) MCF 10A (A), MDA-MB-231 (B), and MCF7 (C) cells transiently expressing GFP-GOLPH3 were held in a microscope stage at 37°C and examined by fluorescence microscopy. The time after initiation of imaging is shown in the bottom right corner of each panel in minutes:seconds. Images are representative of 15–20 videos of up to 200 seconds of recording. In B, filled arrows indicate a vesicular structure moving from the Golgi to the periphery of the cell. In C, filled arrows indicate a vesicular structure moving from the periphery of the cell to the Golgi area, and filled arrowheads indicate a tubular structure elongating from the Golgi. Empty arrows and empty arrowheads indicate the initial position of mobile structures. Bars, 5 μm. (D-F) The number of tubule-vesicular structures moving centrifugally (D), the number of tubular structures elongating from the Golgi (E), or the number of tubule-vesicular structures moving centripetally (F), were quantified from videos corresponding to 180 seconds of imaging. Bar represents the mean + standard deviation of the observed profiles (n = 15). * P < 0.05; *** P < 0.001.

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Expressing, Microscopy, Fluorescence, Imaging, Standard Deviation

Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: (A-C) MCF 10A (A), MDA-MB-231 (B), and MCF7 (C) cells transiently expressing GFP-GOLPH3 were held in a microscope stage at 37°C. The area indicated by a white dotted-line rectangle in each set of images was bleached with a 488-nm laser set to 100% power. The fluorescence recovery after photobleaching (FRAP) was tracked by laser confocal microscopy with the 488-nm laser set to 2% power. Images were acquired before bleaching ( Pre-bleaching ), immediately after bleaching ( Bleaching ), and during the recovery of the fluorescence ( Recovery ) at approximately every 0.4-sec. Images of a representative experiment performed on each cell line are shown in each set of panels. Two images of the recovery of fluorescence are depicted with the time indicated in parenthesis in seconds. Bar, 10 μm. (D) Plot of the FRAP analysis of GFP-GOLPH3 in MCF 10A (black circles; n = 10), MDA-MB-231 (white circles; n = 10), and MCF7 (white squares; n = 10) cells. P , pre-bleaching; B ; bleaching. For simplicity, error bars are not depicted. *** P < 0.001; ns , not statistically significant. The halftime ( t 1/2 ) of maximal fluorescence recovery is indicated on the right in seconds (s).

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Expressing, Microscopy, Fluorescence, Confocal Microscopy

Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: Samples (30 μg of proteins) of rat liver cytosol ( Cyt ), rat liver Golgi membranes, and of cytosolic ( Cyt ) and membrane ( Memb ) fractions from the cell lines indicated at the right were analyzed by two-dimensional gel electrophoresis (2-D GE) and immunoblotting using antibody to GOLPH3. Samples of rat liver Golgi membranes, and of the cytosolic and membrane fractions of each cell line, were dephosphorylated with calf intestine alkaline phosphatase ( CIAP ) before processing for 2-D GE. The position of molecular mass markers is indicated on the left. The position of isoelectric point ( pI ) markers is indicated at the bottom. Red asterisks indicate the position of additional, less abundant, but distinct spots in the samples of MCF7 cells that have slightly slower electrophoretic mobility. Numbers indicate different acidic forms identified in immunoblot films subjected to different exposure times.

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Membrane, Two-Dimensional Gel Electrophoresis, Electrophoresis, Western Blot

Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: (A) Membranes with the spotted phospholipids indicated on the left were incubated with untreated, recombinant GOLPH3 ( GOLPH3 ) or with recombinant GOLPH3 in the presence of cytosolic proteins from the cell lines indicated on the top. Bound recombinant GOLPH3 was detected by immunoblotting with antibody to GOLPH3. LysoPtdA , lysophosphatidic acid; LysoPtdCho , lysophosphatidylcholine; PtdIns , phosphatidylinositol; PtdIns(3)P , phosphatidylinositol 3-phosphate; PtdIns(4)P , phosphatidylinositol 4-phosphate; PtdIns(5)P , phosphatidylinositol 5-phosphate; PtdEth , phosphatidylethanolamine; PtdCho , phosphatidylcholine; S1P , sphingosine 1-phosphate; PtdIns(3 , 4)P 2 , phosphatidylinositol 3,4-bisphosphate; PtdIns(3 , 5)P 2 , phosphatidylinositol 3,5-bisphosphate; PtdIns(4 , 5)P 2 , phosphatidylinositol 4,5-bisphosphate; PtdIns(3 , 4 , 5)P 3 , phosphatidylinositol 3,4,5-trisphosphate; PtdA , phosphatidic acid; PtdSer , phosphatidylserine; Blank , no lipid. (B) Densitometric quantification of the immunoblot signal of the levels of untreated, recombinant GOLPH3 bound to different phospholipids as shown in (A). (C) Densitometric quantification of the immunoblot signal of the levels of recombinant GOLPH3 bound to phosphatidylinositol 4-phosphate after incubation with cytosolic proteins of the indicated cell lines as shown in (A). * P < 0.05; *** P < 0.001.

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Incubation, Recombinant, Western Blot

Journal:

Article Title: Mobilization of Protein Kinase C in Macrophages Induced by Listeria monocytogenes Affects Its Internalization and Escape from the Phagosome

doi: 10.1128/IAI.70.8.4650-4660.2002

Figure Lengend Snippet: Translocation of PKC β II to early endosomes of host J774 cells after infection with L. monocytogenes. PKC β II was labeled with FITC-conjugated secondary antibody as described in the text, and cells labeled for PKC β II are shown in the left panel of each row. Early endosomes labeled with rhodamine-conjugated transferrin are shown in the middle panel of each row. In uninfected cells these endosomes appear as discrete red areas near the periphery of a cell. In uninfected cells PKC β II is distributed throughout the cells and is also colocalized with early endosomes in ≤10% of the cells. The right panel of each row is the merged left and middle panels obtained by using the Phase 3 Imaging software. Upon infection with the wild type, PKC β II translocation to the early endosomes 45 s after addition of the bacteria appears as bright, discrete yellow areas in the same location as the early endosomes (f). PKC β II translocation did not occur after infection with mutant strains lacking LLO (g to i) or PI-PLC (j to l).

Article Snippet: In experiments in which PKC β colocalization to early endosomes was studied, J774 cells were also labeled with rhodamine-conjugated transferrin (Molecular Probes), a specific marker for early endosomes.

Techniques: Translocation Assay, Infection, Labeling, Imaging, Software, Mutagenesis

Journal:

Article Title: Mobilization of Protein Kinase C in Macrophages Induced by Listeria monocytogenes Affects Its Internalization and Escape from the Phagosome

doi: 10.1128/IAI.70.8.4650-4660.2002

Figure Lengend Snippet: PKC β I translocation occurred after infection with the wild type or the PI-PLC mutant strain but not after infection with the LLO mutant strain. PKC β I was labeled with FITC-conjugated secondary antibody as described in the text, and cells labeled for PKC β I are shown in the left panel of each row. Early endosomes were labeled with rhodamine-conjugated transferrin and appear in the middle panel of each row as discrete red areas. In uninfected cells PKC β I was located throughout the cytosol, could also appear as a discrete green area around the periphery of a cell, and could be colocalized with early endosomes. Cells were infected with the strains for 4 min. PKC β I translocation after infection with the wild-type strain (d to f) or the PI-PLC mutant strain (g to i) appears as discrete bright yellow areas corresponding to PKC β I colocalization with transferrin-labeled early endosomes. The images in the right panels were obtained as described in the legend to Fig. ​Fig.2.2. Cells infected with the strain lacking LLO did not show translocation of PKC β I to early endosomes (j to l). Bright green objects seen especially in panels j and l are FITC-labeled bacteria.

Article Snippet: In experiments in which PKC β colocalization to early endosomes was studied, J774 cells were also labeled with rhodamine-conjugated transferrin (Molecular Probes), a specific marker for early endosomes.

Techniques: Translocation Assay, Infection, Mutagenesis, Labeling

Journal:

Article Title: Mobilization of Protein Kinase C in Macrophages Induced by Listeria monocytogenes Affects Its Internalization and Escape from the Phagosome

doi: 10.1128/IAI.70.8.4650-4660.2002

Figure Lengend Snippet: Time course of translocation of PKC β I to early endosomes. Cells were infected with the wild type and harvested at intervals after infection. Early endosomes were isolated, and proteins were prepared for SDS-polyacrylamide gel electrophoresis and Western blotting as described in Materials and Methods. The proteins were also blotted with antibody to the transferrin receptor (upper bands). Infections were carried out for 1 min (lane 1), 3 min (lane 2), 4 min (lane 3), and 5 min (lane 4).

Article Snippet: In experiments in which PKC β colocalization to early endosomes was studied, J774 cells were also labeled with rhodamine-conjugated transferrin (Molecular Probes), a specific marker for early endosomes.

Techniques: Translocation Assay, Infection, Isolation, Polyacrylamide Gel Electrophoresis, Western Blot