Journal: PLoS ONE

Article Title: Characterization of the Mitochondrial Localization of the Nuclear Receptor SHP and Regulation of Its Subcellular Distribution by Interaction with Bcl2 and HNF4α

doi: 10.1371/journal.pone.0068491

Figure Lengend Snippet: (A) The subcellular localization of GFP-hSHP in 293T cells was observed under a fluorescence microscope, and HNF4α was visualized by immunofluorescence using a specific HNF4α antibody. Scale bars: 100 µm, 50 µm (insets). (B) The N-terminal domain of SHP is primarily responsible for the interaction with HNF4α protein. Left, Diagram showing the deletion mutation constructs of GFP-mShp. Right, 293T cells were transfected with GFP-mSHP deletion constructs (4 µg, 6 cm plate) along with HA-HNF4α (4 µg, 6 cm plate) expression vector. Anti-HA agarose was used to immuneprecipitate HNF4α, and the protein levels of various GFP-mSHP truncation mutants and HNF4α were detected by Western blots using anti-GFP or anti-HA antibodies, respectively. Abbreviations: FL, full length; N, N-terminal domain; Int, interaction domain; Rep, repression domain.

Article Snippet: The VDAC antibody (#4661), COX IV antibody (#4850), Hsp60 antibody (#4870), Cyto c antibody (#4280), PARP antibody (#9532), Bcl2 antibody (#2870), myc antibody (#2278), GFP antibody (#2955), GST antibody(#2625), and HNF4α antibody (#3113) were purchased from Cell Signaling.

Techniques: Fluorescence, Microscopy, Immunofluorescence, Mutagenesis, Construct, Transfection, Expressing, Plasmid Preparation, Western Blot

Journal: PLoS ONE

Article Title: Characterization of the Mitochondrial Localization of the Nuclear Receptor SHP and Regulation of Its Subcellular Distribution by Interaction with Bcl2 and HNF4α

doi: 10.1371/journal.pone.0068491

Figure Lengend Snippet: The subcellular localization of GFP-mSHP full length and deletion mutants in 293T cells was observed by fluorescence microscopy, and HNF4α was visualized by immunofluorescence using a specific HNF4α antibody. Scale bars: 50 µm. Abbreviations: FL, full length; N, N-terminal domain; Int, interaction domain; Rep, repression domain.

Article Snippet: The VDAC antibody (#4661), COX IV antibody (#4850), Hsp60 antibody (#4870), Cyto c antibody (#4280), PARP antibody (#9532), Bcl2 antibody (#2870), myc antibody (#2278), GFP antibody (#2955), GST antibody(#2625), and HNF4α antibody (#3113) were purchased from Cell Signaling.

Techniques: Fluorescence, Microscopy, Immunofluorescence

Journal: PLoS ONE

Article Title: Retigeric Acid B Attenuates the Virulence of Candida albicans via Inhibiting Adenylyl Cyclase Activity Targeted by Enhanced Farnesol Production

doi: 10.1371/journal.pone.0041624

Figure Lengend Snippet: (A) Nematodes were infected with C. albicans YEM30 for 2 h and then moved to pathogen-free liquid media in the presence of PBS (negative control), RAB (4 or 16 µg/ml) or 2 µg/ml of FLC (positive control). Each day the worms were monitored and the survival rate was calculated. p <0.001 for 4RAB, 16RAB and FLC treated groups compared to PBS-treated group. (B) Nematodes were infected with the indicated C. albicans strains and then treated by a series of doses of RAB. After 5 days of therapy, the survival ratio in each group was calculated. (C) The nematodes were infected by C. albicans strain CASA1 with GFP tagged CDR1 for 2 h and then moved into pathogen-free liquid media containing drugs or the vehicle for 5 days. And then they were observed in 4× objective magnification. Most nematodes in control group did not demonstrate any movement and developed filaments outside the body. However, the majority of the drug-treated were alive and not invaded by the hyphae (scale bars: 200 µm). Insert shows the images of nematodes captured at 200× magnification (scale bars: 100 µm). The representative worm was selected for confocal microscopic observation. Green fluorescence of hyphae cells and yeast cells was seen within the intestine of the dead nematode under the vehicle treatment. And no C. albicans cells were observed in the live worm challenged by RAB (about 60 nematodes in each group). (D) Enlarged view of the rectangle frame.

Article Snippet: Subsequently, the fixed cells were washed three times with PBS and incubated with mouse anti-GFP monoclonal antibody (Cell Signaling Technology, Beverly, MA) diluted 1∶200 in PBS with 1% BSA for 1 h at room temperature.

Techniques: Infection, Negative Control, Positive Control, Fluorescence

Journal: PLoS ONE

Article Title: E1-Like Activating Enzyme Atg7 Is Preferentially Sequestered into p62 Aggregates via Its Interaction with LC3-I

doi: 10.1371/journal.pone.0073229

Figure Lengend Snippet: (A) Expression plasmids were transfected into HEK293 cells for 24 h. Cell lysates were used to detect the fusion genes’ expression by Western blot using antibodies as indicated. (B) Flag-LC3 or Flag-LC3 A120 was transfected into HEK293 cells for 24 h. 50 µM of Chloroquine (CQ) was used to induce Flag- LC3-II accumulation for 2 h before harvesting the cells. Flag-LC3 is normally lipidated as demonstrated by Flag-LC3-II accumulation after CQ treatment. No such accumulation was seen in Flag-LC3 A120 mutant after CQ treatment demonstrating that this mutant does not pass through the lipidation process. (C) MEFwt cells were transfected with indicated expression plasmids for 24 h before they were recorded by fluorescence microscopy. GFP-Atg4B, GFP-Atg7 and GFP-Atg3 were mainly distributed in the cytosol. No puncta was observed at any given time. Scale bar = 25micron.

Article Snippet: The following antibodies were used: rabbit polyclonal anti-LC3B, Atg4B, Atg3, and Atg7 antibodies (Cell Signaling); mouse monoclonal anti-GFP and p62 antibodies (Santa Cruz biotech); mouse monoclonal anti-Flag M2 and β-actin antibodies (Sigma); GFP-Trap (Allele Biotechnology); anti-rabbit IgG (Alexa Fluor 488 conjugate) and anti-mouse IgG (Alexa Fluor 555 conjugate) (Cell signaling); anti-mouse IgG(AMCA-conjugate) (Jackson ImmunoResearch).

Techniques: Expressing, Transfection, Western Blot, Mutagenesis, Fluorescence, Microscopy

Journal: PLoS ONE

Article Title: E1-Like Activating Enzyme Atg7 Is Preferentially Sequestered into p62 Aggregates via Its Interaction with LC3-I

doi: 10.1371/journal.pone.0073229

Figure Lengend Snippet: GFP-Atg4B and mRFP-p62 expression plasmids (A) or with Flag-LC3 (B) were co-transfected into MEFwt cells for 24h. GFP-Atg4B C74A and mRFP-p62 with Flag-LC3 vectors (C) or with Flag-LC3 A120 vector (D) were co-transfected into MEFwt cells for 24 h. GFP-Atg4B or GFP-Atg4B C74A expression vector was co-transfected with Flag-LC3 (E) or Flag-LC3 A120 (F) into HEK293 cells for 48 h. Immunoprecipitation (IP) was performed using total cell lysates. Immunoblotting was conducted using the antibodies as indicated. (G) mRFP-p62, GFP-Atg4B C74A and Flag-LC3 expression vectors were co-transfected into MEFwt cells for 24 h. Colocalization of Flag-LC3 within mRFP-p62 aggregates were detected by immunofluorescence staining (IF) using Flag M2 antibody. Fluorescence images presented in this study were representative of at least three independent experiments. Scale bar = 25micron.

Article Snippet: The following antibodies were used: rabbit polyclonal anti-LC3B, Atg4B, Atg3, and Atg7 antibodies (Cell Signaling); mouse monoclonal anti-GFP and p62 antibodies (Santa Cruz biotech); mouse monoclonal anti-Flag M2 and β-actin antibodies (Sigma); GFP-Trap (Allele Biotechnology); anti-rabbit IgG (Alexa Fluor 488 conjugate) and anti-mouse IgG (Alexa Fluor 555 conjugate) (Cell signaling); anti-mouse IgG(AMCA-conjugate) (Jackson ImmunoResearch).

Techniques: Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Immunofluorescence, Staining, Fluorescence

Journal: PLoS ONE

Article Title: E1-Like Activating Enzyme Atg7 Is Preferentially Sequestered into p62 Aggregates via Its Interaction with LC3-I

doi: 10.1371/journal.pone.0073229

Figure Lengend Snippet: Vectors expressing mRFP-p62, GFP-LC3, or GFP-LC3 A120 were transfected into MEFatg5 −/− cells as indicated (A and B). Images were taken at 48 h time point. The same sets of plasmids were transfected into MEFwt cells as indicated for 72 h. Images were recorded at 20 h and 72 h post transfection (C–F). Scale bar = 25micro. Arrows point to mRFP-p62 only punctae. The inserts were enlarged to show the puncta in detail.

Article Snippet: The following antibodies were used: rabbit polyclonal anti-LC3B, Atg4B, Atg3, and Atg7 antibodies (Cell Signaling); mouse monoclonal anti-GFP and p62 antibodies (Santa Cruz biotech); mouse monoclonal anti-Flag M2 and β-actin antibodies (Sigma); GFP-Trap (Allele Biotechnology); anti-rabbit IgG (Alexa Fluor 488 conjugate) and anti-mouse IgG (Alexa Fluor 555 conjugate) (Cell signaling); anti-mouse IgG(AMCA-conjugate) (Jackson ImmunoResearch).

Techniques: Expressing, Transfection

Journal: PLoS ONE

Article Title: E1-Like Activating Enzyme Atg7 Is Preferentially Sequestered into p62 Aggregates via Its Interaction with LC3-I

doi: 10.1371/journal.pone.0073229

Figure Lengend Snippet: GFP-Atg7 (Aa), GFP-Atg7/Flag-LC3 (Ab), or GFP-Atg7 C572A /Flag-LC3 (Ac) vectors were co-transfected with mRFP-p62 into MEFwt cells for 48 h. GFP-Atg7 punctae were shown; mRFP-p62 and Flag-LC3 with GFP-Atg7 vectors (B) or with GFP-Atg7 C572A (C) were co-transfected into MEFwt cells for 48 h; mRFP-p62 and Flag-LC3A 120 with GFP-Atg7 vectors (D) or with GFP-Atg7 C572A (E) were co-transfected into MEFwt cells for 48 h. Flag-LC3, GFP-Atg7, or GFP-Atg7 C572A expression vectors were co-transfected into HEK293 cells as indicated for 48 h. Total cell lysates were used for IP by the Flag M2 antibody. Immunoblotting was conducted using the antibodies as indicated (F and G). Expressing vectors of GFP-Atg12 and mRFP-p62 were co-transfected into MEFwt cells for 48 h (H). All images were recorded at 48 h post-transfection. Scale bar = 25 micron. The inserts were enlarged to show the puncta in detail.

Article Snippet: The following antibodies were used: rabbit polyclonal anti-LC3B, Atg4B, Atg3, and Atg7 antibodies (Cell Signaling); mouse monoclonal anti-GFP and p62 antibodies (Santa Cruz biotech); mouse monoclonal anti-Flag M2 and β-actin antibodies (Sigma); GFP-Trap (Allele Biotechnology); anti-rabbit IgG (Alexa Fluor 488 conjugate) and anti-mouse IgG (Alexa Fluor 555 conjugate) (Cell signaling); anti-mouse IgG(AMCA-conjugate) (Jackson ImmunoResearch).

Techniques: Transfection, Expressing, Western Blot

Journal: PLoS ONE

Article Title: E1-Like Activating Enzyme Atg7 Is Preferentially Sequestered into p62 Aggregates via Its Interaction with LC3-I

doi: 10.1371/journal.pone.0073229

Figure Lengend Snippet: GFP-Atg3 and mRFP-p62 (A) or with Flag-LC3 vectors (B) were co-transfected into MEFwt cells for 48 h. GFP-Atg3 C264S , mRFP-p62 and Flag-LC3 vectors were co-transfected into MEFwt cells for 48 h (C). Flag-LC3, GFP-Atg3, GFP-Atg3 C264S , or GFP-Atg3 C264A expression vectors were co-transfected into HEK293 cells for 48 h. Immunoprecipitation was conducted using total cell lysates. Immunoblotting was conducted as indicated. Images were recorded at 48 h post-transfection. Scale bar = 25 micron.

Article Snippet: The following antibodies were used: rabbit polyclonal anti-LC3B, Atg4B, Atg3, and Atg7 antibodies (Cell Signaling); mouse monoclonal anti-GFP and p62 antibodies (Santa Cruz biotech); mouse monoclonal anti-Flag M2 and β-actin antibodies (Sigma); GFP-Trap (Allele Biotechnology); anti-rabbit IgG (Alexa Fluor 488 conjugate) and anti-mouse IgG (Alexa Fluor 555 conjugate) (Cell signaling); anti-mouse IgG(AMCA-conjugate) (Jackson ImmunoResearch).

Techniques: Transfection, Expressing, Immunoprecipitation, Western Blot

Journal: PLoS ONE

Article Title: E1-Like Activating Enzyme Atg7 Is Preferentially Sequestered into p62 Aggregates via Its Interaction with LC3-I

doi: 10.1371/journal.pone.0073229

Figure Lengend Snippet: HEK293 cells were co-transfected with mRFP-p62/GFP-Atg3/Flag-LC3 or mRFP-p62/GFP-Atg4B/Flag-LC3 for 48 h. Total cell lysates were used to conduct IP using correspondent antibodies. Immunoblottings were then applied using antibodies as indicated (A and B). HEK293 cells were transfected with GFP-Atg4B or GFP-Atg4B C74A alone or with mRFP-p62 for 48 h (C). HEK293 cells were transfected with GFP-Atg3 alone or with mRFP-p62 for 48 h (D). Total cell lysates were used to detect LC3 species as indicated. Relative band intensity of protein of interest was divided by correspondent band intensity of β-actin loading control. Fold of increase or decrease was then determined by setting the control sample as 1.

Article Snippet: The following antibodies were used: rabbit polyclonal anti-LC3B, Atg4B, Atg3, and Atg7 antibodies (Cell Signaling); mouse monoclonal anti-GFP and p62 antibodies (Santa Cruz biotech); mouse monoclonal anti-Flag M2 and β-actin antibodies (Sigma); GFP-Trap (Allele Biotechnology); anti-rabbit IgG (Alexa Fluor 488 conjugate) and anti-mouse IgG (Alexa Fluor 555 conjugate) (Cell signaling); anti-mouse IgG(AMCA-conjugate) (Jackson ImmunoResearch).

Techniques: Transfection

Journal: PLoS ONE

Article Title: E1-Like Activating Enzyme Atg7 Is Preferentially Sequestered into p62 Aggregates via Its Interaction with LC3-I

doi: 10.1371/journal.pone.0073229

Figure Lengend Snippet: HEK293-GFP-LC3 cells were cultured in nutrient-rich media (Aa) or treated with CPP for 2 h (Ab) to induce large GFP-LC3 positive vesicles. HEK293-GFP-LC3 A120 cells were used as a control (Ac); (B) HEK293-GFP-LC3 cells were transfected with mRFP-p62 for 24 h, and then treated with CPP for 2 h to induce large GFP-LC3/mRFP-p62 positive vesicles. (C) HEK293-GFP-LC3 cells were transfected with mRFP-p62-LRS-mut.2 for 24 h, and then treated with CPP for 2 h. HEK293-GFP-LC3 cell line was cultured in regular media (D) or treated with CPP for 2 h (E) before fixed and immunostained for the endogenous LC3 and p62. (F) HEK293-GFP-LC3 cells were cultured in regular media or treated with CPP for 2 h. Total cell lysates were used for immunoprecipitation of endogenous p62. Western blot analysis was conducted to detect p62 bound GFP-LC3 species. Arrow points to GFP-LC3 positive vesicle. Arrow head points to p62 aggregate. Scale bar = 25 micron.

Article Snippet: The following antibodies were used: rabbit polyclonal anti-LC3B, Atg4B, Atg3, and Atg7 antibodies (Cell Signaling); mouse monoclonal anti-GFP and p62 antibodies (Santa Cruz biotech); mouse monoclonal anti-Flag M2 and β-actin antibodies (Sigma); GFP-Trap (Allele Biotechnology); anti-rabbit IgG (Alexa Fluor 488 conjugate) and anti-mouse IgG (Alexa Fluor 555 conjugate) (Cell signaling); anti-mouse IgG(AMCA-conjugate) (Jackson ImmunoResearch).

Techniques: Cell Culture, Transfection, Immunoprecipitation, Western Blot

Journal: PLoS ONE

Article Title: Concerted Action of the Ubiquitin-Fusion Degradation Protein 1 (Ufd1) and Sumo-Targeted Ubiquitin Ligases (STUbLs) in the DNA-Damage Response

doi: 10.1371/journal.pone.0080442

Figure Lengend Snippet: (A) Yeast two-hybrid interactions. 10-fold dilution series of S . cerevisae strain PJ69-4A expressing a C-terminal domain of Ufd1 (aa 248-342) fused to the Gal4 DNA activation domain (GAD) together with either Pli1 or Rfp1 fused to the Gal4 DNA-binding domain (GBD) were spotted on the indicated media. Protein interactions result in activation of the ADE2 reporter gene in the tester strain and growth on SC-leu-trp-ade. -: empty vectors. (B) Ufd1Ct interacts with Pli1 and Rfp1 in vitro . GST pull-down experiment with GST, GST- Pli1 or GST-Rfp1 incubated with in vitro -translated, 35 S-labeled Ufd1 (aa 110-342). (C) Summary of two-hybrid interactions. Red arrows indicate novel interactions identified in this study; black arrows, previously reported interactions that were (solid arrows) or not (dashed arrows) also identified in our screens. (D) Co-purification of Cdc48 and Slx8. Cdc48-GFP was purified on a GFP affinity matrix from cells expressing Myc-tagged Slx8 or an untagged control. Cdc48-GFP and Slx8-Myc were detected by Western blotting with respectively GFP and Myc antibodies. (E) Accumulation of sumoylated proteins in ufd1ΔCt 213-342 cells. Whole-cell extracts of wild type and indicated mutants were probed with an anti-SUMO antibody. The slx8-1 and mts3-1 mutants were shifted from 30°C to 37°C for 4 hr prior to harvesting; all other strains were propagated at 30°C. The strains were, from left to right, JK8; JK9; JK10; JK11; NBY1008; PI131; Δsph2.

Article Snippet: Cdc48-GFP was identified by the anti-GFP mouse antibody from Roche and co-precipitated Slx8-Myc was identified with a Myc-tag mouse antibody from Cell Signaling Technology.

Techniques: Expressing, Activation Assay, Binding Assay, In Vitro, Incubation, Labeling, Copurification, Purification, Western Blot

Journal: PLoS ONE

Article Title: Concerted Action of the Ubiquitin-Fusion Degradation Protein 1 (Ufd1) and Sumo-Targeted Ubiquitin Ligases (STUbLs) in the DNA-Damage Response

doi: 10.1371/journal.pone.0080442

Figure Lengend Snippet: (A) ufd1ΔCt 213-342 cells are sensitive to DNA damage: 10-fold dilution series of the indicated strains were spotted onto YES plates containing camptothecin (CPT) or hydroxyurea (HU), or subsequently exposed to UV irradiation as indicated. (B) Fluorescence imaging of wild-type and ufd1ΔCt 213-342 asynchronous cultures expressing Rad22-YFP. (C) ufd1ΔCt 213-342 cells display an increased frequency of spontaneous Rad22 foci: Nuclei were classified according to their position in the cell cycle based on cell morphology. Bars represent the percentage of nuclei with at least one Rad22-YFP focus averaged from three independent experiments. Error bars correspond to the standard deviations for the combined data and p-values were calculated with a Fishers exact test. More than 300 cells of each strain were counted in each experiment, the numbers are reported in . Only very few cells were counted in the ‘multiple nuclei/septa’ category, giving rise to the large standard deviations in this category. (D) ufd1ΔCt 213-342 cells are unable to recover from Zeocin-induced damage: Rad22-YFP foci were quantified after 1 hr of growth in Zeocin-containing medium (300 µg/ml) and again 13 hr after Zeocin had been removed from the media. Between 50 and 100 cells were counted for each strain at each time point; error bars indicate exact binomial 95% confidence intervals. Indicated p-values were obtained with a Fishers exact test. Fluorescence images are shown in . (E) and (F) Rad22-YFP accumulation in ufd1ΔCt 213-342 cells: (E) Rad22-YFP from whole cell extracts of the indicated strains was detected by an anti-GFP antibody. (F) Rad22-YFP was affinity-purified from a wild-type or ufd1ΔCt 213-342 genetic background and detected by anti-GFP immunoblotting. HMW indicates higher molecular weight species of Rad22-YFP accumulating in ufd1ΔCt 213-342 . * indicates a crossreacting species.

Article Snippet: Cdc48-GFP was identified by the anti-GFP mouse antibody from Roche and co-precipitated Slx8-Myc was identified with a Myc-tag mouse antibody from Cell Signaling Technology.

Techniques: Irradiation, Fluorescence, Imaging, Expressing, Affinity Purification, Western Blot, Molecular Weight