Journal:

Article Title: Modulation of Gamma Interferon-Induced Major Histocompatibility Complex Class II Gene Expression by Porphyromonas gingivalis Membrane Vesicles

doi: 10.1128/IAI.70.3.1185-1192.2002

Figure Lengend Snippet: Inhibition of class II HLA-DRα surface expression in IFN-γ-inducible cells by P. gingivalis membrane vesicles. HUVECs, 143B osteosarcoma cells, MRC-5 fibroblasts, and THP-1 monocytic cells were treated with vesicles (OMVs, 30 μg of protein/ml) alone or IFN-γ (250 U/ml) in the absence or presence of vesicles at 37°C for 3 days. Control cells received no treatment. Cells were stained with either phycoerythrin-conjugated mouse anti-HLA-DR MAb or irrelevant isotype-matched MAb. Stained cells were analyzed by flow cytometry. Averages and standard deviations were calculated from three separate experiments.

Article Snippet: Other cell lines used, obtained from ATCC, included human embryonic lung fibroblasts (MRC-5; ATCC CCL 171), 143B human osteosarcoma cells (ATCC CRL 8304), and THP-1 monocytic cells (ATCC TIB 202).

Techniques: Inhibition, Expressing, Membrane, Control, Staining, Flow Cytometry

Journal: Life Science Alliance

Article Title: Lipid-associated PML structures assemble nuclear lipid droplets containing CCTα and Lipin1

doi: 10.26508/lsa.202000751

Figure Lengend Snippet: U2OS and PML KO cells were cultured in 400 μM oleate for 24 h before fixation and immunostaining for CCTα (blue) and PML (red) and imaging by confocal microscopy. LDs were visualized with BODIPY 493/503 (green) (bar, 10 μm). Arrows indicate the region selection for RGB line scan plots showing the co-localization of PML and CCTα on the surface of nuclear LDs and the nuclear envelope.

Article Snippet: Cells were lysed in SDS buffer (12.5% SDS, 30 mm Tris–HCl, 12.5% glycerol, and 0.01% bromophenol blue [pH 6.8]) and heated at 95°C for 5 min. Lysates were separated by SDS–PAGE, transferred to nitrocellulose membranes, and incubated in TBS (20 mM Tris–HCl [pH 7.4] and 500 mM NaCl):Odyssey Blocking Buffer (5:1, vol/vol) for 1 h. Nitrocellulose was blotted using primary antibodies against human CCTα , PML (rabbit polyclonal A301–167A; Bethyl Laboratories), V5 monoclonal (MCA-1360; Bio-Rad), or β-actin (mouse monoclonal AC15; Sigma-Aldrich).

Techniques: Cell Culture, Immunostaining, Imaging, Confocal Microscopy, Selection

Journal: Life Science Alliance

Article Title: Lipid-associated PML structures assemble nuclear lipid droplets containing CCTα and Lipin1

doi: 10.26508/lsa.202000751

Figure Lengend Snippet: (A) Lysates of U2OS and PML KO cells that were untreated or incubated with oleate (400 μM) for 24 h were immunoblotted with PML-, CCTα-, and actin-specific antibodies. (B) U2OS and PML KO cells were immunostained for CCTα and LMNA/C, and LDs were visualized with BODIPY 493/503 (bar, 20 μm). Arrows in magnified panels (a) and (b) indicate the regions for RGB line scan plots showing the localization of CCTα on the surface of nLDs. (C, D) Quantitation of cytoplasmic (cLDs) and nLDs in cells treated with oleate (400 μM) for 24 h. (E) Quantitation of CCTα-positive nLDs in oleate-treated cells. (F, G) The size distribution of cLDs (panel F) and nLDs (panel G) in oleate-treated cells was determined by cross-sectional area binning. (H) Average cross-sectional area of total and CCTα-positive nLDs was quantified in oleate-treated cells. In panels (C, D, E, F, G, H), results are presented as box and whisker plots showing the mean and 5 th –95 th percentile for analysis of 50–100 cells from three separate experiments. Significance was determined by two-tailed t test compared with matched U2OS cell controls (** P < 0.01; *** P < 0.001).

Article Snippet: Cells were lysed in SDS buffer (12.5% SDS, 30 mm Tris–HCl, 12.5% glycerol, and 0.01% bromophenol blue [pH 6.8]) and heated at 95°C for 5 min. Lysates were separated by SDS–PAGE, transferred to nitrocellulose membranes, and incubated in TBS (20 mM Tris–HCl [pH 7.4] and 500 mM NaCl):Odyssey Blocking Buffer (5:1, vol/vol) for 1 h. Nitrocellulose was blotted using primary antibodies against human CCTα , PML (rabbit polyclonal A301–167A; Bethyl Laboratories), V5 monoclonal (MCA-1360; Bio-Rad), or β-actin (mouse monoclonal AC15; Sigma-Aldrich).

Techniques: Incubation, Quantitation Assay, Whisker Assay, Two Tailed Test

Journal: Life Science Alliance

Article Title: Lipid-associated PML structures assemble nuclear lipid droplets containing CCTα and Lipin1

doi: 10.26508/lsa.202000751

Figure Lengend Snippet: (A) 3D surface reconstruction of image stacks of oleate-treated (400 μM for 24 h) U2OS cells immunostained for PML and CCTα. LDs and nuclei were visualized with BODIPY 493/503 and DAPI, respectively. A large nLD was identified in association with CCTα and PML (panel a, red frame). The image was rotated and zoomed to produce a side view of the nLD (panel b, red frame). The framed structure in (b) was assessed by removing the DAPI channel (panels c–f) to reveal how the nLD/BODIPY (blue), CCTα (green), and PML (red) are associated. (A, B) Wide-field images of the same nLD in (A) imaged at the level of the yellow line indicated in panels b–f (image rotated ∼90° clockwise). The nLD is highlighted and magnified in the inset (bar, 5 μm; inset scale bar, 1 μm). (B, C) Images of the same field of view in (B) were acquired by NanoJ SRRF. The nLD is highlighted and magnified in the inset (bar, 5 μm; inset bar, 1 μm).

Article Snippet: Cells were lysed in SDS buffer (12.5% SDS, 30 mm Tris–HCl, 12.5% glycerol, and 0.01% bromophenol blue [pH 6.8]) and heated at 95°C for 5 min. Lysates were separated by SDS–PAGE, transferred to nitrocellulose membranes, and incubated in TBS (20 mM Tris–HCl [pH 7.4] and 500 mM NaCl):Odyssey Blocking Buffer (5:1, vol/vol) for 1 h. Nitrocellulose was blotted using primary antibodies against human CCTα , PML (rabbit polyclonal A301–167A; Bethyl Laboratories), V5 monoclonal (MCA-1360; Bio-Rad), or β-actin (mouse monoclonal AC15; Sigma-Aldrich).

Techniques:

Journal: Life Science Alliance

Article Title: Lipid-associated PML structures assemble nuclear lipid droplets containing CCTα and Lipin1

doi: 10.26508/lsa.202000751

Figure Lengend Snippet: The original SRRF image before cropping from is shown for DAPI, PML (AlexaFluor-647), BODIPY 493/503, and CCTα (AlexaFluor-568) fluorescent micrographs in the top row of each image grouping. The middle row in each grouping represents the resolution estimate divided in ∼750 blocks by Fourier ring correlation (FRC) analysis (mean estimated resolution is shown in the inset at the left). The bottom row represents an Error Map based on the resolution scale Pearson (RSP) correlation and the resolution scale error (RSE).

Article Snippet: Cells were lysed in SDS buffer (12.5% SDS, 30 mm Tris–HCl, 12.5% glycerol, and 0.01% bromophenol blue [pH 6.8]) and heated at 95°C for 5 min. Lysates were separated by SDS–PAGE, transferred to nitrocellulose membranes, and incubated in TBS (20 mM Tris–HCl [pH 7.4] and 500 mM NaCl):Odyssey Blocking Buffer (5:1, vol/vol) for 1 h. Nitrocellulose was blotted using primary antibodies against human CCTα , PML (rabbit polyclonal A301–167A; Bethyl Laboratories), V5 monoclonal (MCA-1360; Bio-Rad), or β-actin (mouse monoclonal AC15; Sigma-Aldrich).

Techniques:

Journal: Life Science Alliance

Article Title: Lipid-associated PML structures assemble nuclear lipid droplets containing CCTα and Lipin1

doi: 10.26508/lsa.202000751

Figure Lengend Snippet: U2OS cells were treated with oleate for 24 h before fixation and immunostaining for CCTα (green) and PML (red). nLDs were localized based on BODIPY 493/503 as described in the legend to . (A, B, C) Spinning disk confocal sections and full 3D rendering (SlideBook) are shown for three representative nuclei (panels A, B, C), and eight nLDs are enlarged (objects 1–6) to better show the relationship between PML and CCTα on nLDs. DNA was visualized with DAPI. The scale bar on confocal images is 10 μm (inset is 2 μm), and the grid on the 3D render is 5 μm.

Article Snippet: Cells were lysed in SDS buffer (12.5% SDS, 30 mm Tris–HCl, 12.5% glycerol, and 0.01% bromophenol blue [pH 6.8]) and heated at 95°C for 5 min. Lysates were separated by SDS–PAGE, transferred to nitrocellulose membranes, and incubated in TBS (20 mM Tris–HCl [pH 7.4] and 500 mM NaCl):Odyssey Blocking Buffer (5:1, vol/vol) for 1 h. Nitrocellulose was blotted using primary antibodies against human CCTα , PML (rabbit polyclonal A301–167A; Bethyl Laboratories), V5 monoclonal (MCA-1360; Bio-Rad), or β-actin (mouse monoclonal AC15; Sigma-Aldrich).

Techniques: Immunostaining

Journal: Life Science Alliance

Article Title: Lipid-associated PML structures assemble nuclear lipid droplets containing CCTα and Lipin1

doi: 10.26508/lsa.202000751

Figure Lengend Snippet: (A) U2OS and PML KO cells transiently expressing a nuclear nGFP-DAG sensor were exposed to 400 μM oleate for 24 h, fixed, and immunostained with a CCTα antibody and incubated with LipidTox Red to visualize LDs (bar, 10 μm). Arrows in the merged image indicate the region selected for an RGB line scan plot showing the association of CCTα and DAG-GFP. (B) Quantification of nLDs in oleate-treated U2OS and PML KO cells containing neither nGFP-DAG or CCTα (CCTα/DAG-GFP-), nGFP-DAG (DAG+), CCTα (CCT+), or both (CCT/DAG+). A Venn diagram shows the percent distribution of nGFP-DAG and CCTα-positive nLDs. (C) In oleate-treated U2OS and PML KO cells, the cross-sectional area of nLDs containing nGFP-DAG and/or CCTα was measured. (B, C) Results are presented as box and whisker plots showing the mean and 5 th –95 th percentile for analysis of 50–100 cells from three separate experiments. Significance was determined by a two-tailed t test compared with matched U2OS controls (** P < 0.01; *** P < 0.001).

Article Snippet: Cells were lysed in SDS buffer (12.5% SDS, 30 mm Tris–HCl, 12.5% glycerol, and 0.01% bromophenol blue [pH 6.8]) and heated at 95°C for 5 min. Lysates were separated by SDS–PAGE, transferred to nitrocellulose membranes, and incubated in TBS (20 mM Tris–HCl [pH 7.4] and 500 mM NaCl):Odyssey Blocking Buffer (5:1, vol/vol) for 1 h. Nitrocellulose was blotted using primary antibodies against human CCTα , PML (rabbit polyclonal A301–167A; Bethyl Laboratories), V5 monoclonal (MCA-1360; Bio-Rad), or β-actin (mouse monoclonal AC15; Sigma-Aldrich).

Techniques: Expressing, Incubation, Whisker Assay, Two Tailed Test

Journal: Life Science Alliance

Article Title: Lipid-associated PML structures assemble nuclear lipid droplets containing CCTα and Lipin1

doi: 10.26508/lsa.202000751

Figure Lengend Snippet: (A) U2OS cells transiently expressing nuclear localized GFP-C1(2)δ (nGFP-DAG) were exposed to oleate (400 μM) for 24 h and were fixed and immunostained with a PML antibody. LDs were visualized with LipidTox Red (bar, 10 μm). Arrows in the merged image indicate the region selected of an RGB line scan plot showing the localization of DAG-GFP with PML and nLDs. (B) Quantitation of nLDs in oleate-treated U2OS cells containing neither nGFP-DAG or PML (DAG/PML−), nGFP-DAG (DAG+), PML (PML+), or both (DAG/PML+). A Venn diagram shows the percent distribution of nGFP-DAG and PML-positive nLDs. (C) The average cross-sectional area of nLDs in oleate-treated U2OS cells containing nGFP-DAG and/or PML as described above. (D) Quantification of DAG-negative (−) and DAG-negative (+) nLDs in U2OS and PML KO cells. (B, C, D) Results are presented as box and whisker plots showing the mean and 5 th –95 th percentile from analysis of 50–100 cells in three separate experiments. Significance was determined by one-way ANOVA and Tukey’s multiple comparison to total nLD area (panel C) or two-tailed t test compared with U2OS controls (panel D) (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: Cells were lysed in SDS buffer (12.5% SDS, 30 mm Tris–HCl, 12.5% glycerol, and 0.01% bromophenol blue [pH 6.8]) and heated at 95°C for 5 min. Lysates were separated by SDS–PAGE, transferred to nitrocellulose membranes, and incubated in TBS (20 mM Tris–HCl [pH 7.4] and 500 mM NaCl):Odyssey Blocking Buffer (5:1, vol/vol) for 1 h. Nitrocellulose was blotted using primary antibodies against human CCTα , PML (rabbit polyclonal A301–167A; Bethyl Laboratories), V5 monoclonal (MCA-1360; Bio-Rad), or β-actin (mouse monoclonal AC15; Sigma-Aldrich).

Techniques: Expressing, Quantitation Assay, Whisker Assay, Two Tailed Test

Journal: Life Science Alliance

Article Title: Lipid-associated PML structures assemble nuclear lipid droplets containing CCTα and Lipin1

doi: 10.26508/lsa.202000751

Figure Lengend Snippet: (A) U2OS and PML KO cells transiently expressing Lipin1α-V5 were immunostained with a V5 monoclonal antibody, and nuclei were visualized with DAPI (bar, 10 μm). (B) U2OS cells transiently expressing Lipin1α-V5 were treated with 400 μM oleate for 24 h and immunostained with a V5 monoclonal antibody. LDs were visualized with BODIPY 493/503 (bar, 10 μm). Panels (a) and (b) show magnified regions for Lipin1α and merge images. Arrows show the region in panel (b) selected for an RGB line scan plot (panel c) showing Lipin1α on the surface of nLDs. (C) Quantification of nLDs in oleate-treated U2OS cells containing neither nGFP-DAG nor Lipin1α (Lipin/PML−), nGFP-DAG (DAG+), Lipin1α (Lipin+), or both (Lipin/DAG+). A Venn diagram shows the percent distribution of nGFP-DAG and Lipin1α-positive nLDs. (D) The cross-sectional area of nLDs containing differing compositions of nGFP-DAG, and Lipin1α was quantified in oleate-treated U2OS cells. (E) PML KO cells were treated with oleate and immunostained as described above (bar, 10 μm). (F) Quantification of Lipin-negative (−) and Lipin-positive (+) nLDs in U2OS and PML KO cells. (C, D, F) Results are presented as box and whisker plots showing the mean and 5 th –95 th percentile for analysis of 50–100 cells from three separate experiments. (C, D, F) Significance was determined by one-way ANOVA and Tukey’s multiple comparison (panels C and D) or two-tailed t test compared with control U2OS cells (panel F) (*** P < 0.001).

Article Snippet: Cells were lysed in SDS buffer (12.5% SDS, 30 mm Tris–HCl, 12.5% glycerol, and 0.01% bromophenol blue [pH 6.8]) and heated at 95°C for 5 min. Lysates were separated by SDS–PAGE, transferred to nitrocellulose membranes, and incubated in TBS (20 mM Tris–HCl [pH 7.4] and 500 mM NaCl):Odyssey Blocking Buffer (5:1, vol/vol) for 1 h. Nitrocellulose was blotted using primary antibodies against human CCTα , PML (rabbit polyclonal A301–167A; Bethyl Laboratories), V5 monoclonal (MCA-1360; Bio-Rad), or β-actin (mouse monoclonal AC15; Sigma-Aldrich).

Techniques: Expressing, Whisker Assay, Two Tailed Test

Journal: Life Science Alliance

Article Title: Lipid-associated PML structures assemble nuclear lipid droplets containing CCTα and Lipin1

doi: 10.26508/lsa.202000751

Figure Lengend Snippet: (A) U2OS cells transiently expressing Lipin1β-V5 were untreated or incubated with 400 μM oleate for 24 h before fixation and immunostaining with a V5 monoclonal antibody (red) and imaged by confocal microscopy. Selected regions from Lipin1β (a) and merge (b) images of oleate-treated cells show the association of Lipin1β with nLD stained with BODIPY 493/503 (green) (bar, 10 μm). Arrows in panel (b) indicate the region selected for RGB line scan plots (panel c) showing Lipin1β is on the surface of nLD. ( B ) Transiently expressed Lipin1β in oleate-treated PML KO cells (as described in panel A) was not detected on the surface of nLDs (bar, 10 μm).

Article Snippet: Cells were lysed in SDS buffer (12.5% SDS, 30 mm Tris–HCl, 12.5% glycerol, and 0.01% bromophenol blue [pH 6.8]) and heated at 95°C for 5 min. Lysates were separated by SDS–PAGE, transferred to nitrocellulose membranes, and incubated in TBS (20 mM Tris–HCl [pH 7.4] and 500 mM NaCl):Odyssey Blocking Buffer (5:1, vol/vol) for 1 h. Nitrocellulose was blotted using primary antibodies against human CCTα , PML (rabbit polyclonal A301–167A; Bethyl Laboratories), V5 monoclonal (MCA-1360; Bio-Rad), or β-actin (mouse monoclonal AC15; Sigma-Aldrich).

Techniques: Expressing, Incubation, Immunostaining, Confocal Microscopy, Staining

Journal: International journal of biological sciences

Article Title: Crosstalk between PML and p53 in response to TGF-β1: A new mechanism of cardiac fibroblast activation.

doi: 10.7150/ijbs.76214

Figure Lengend Snippet: Figure 5. PML stabilizes p53 by directly binding to p53 in response to TGF-β1. (A) Co-localization of PML and p53 was detected by immunofluorescence analysis in CFs after treatment with TGF-β1. Scale bar, 5 μm (n = 5). (B and C) Representative immunoblots showing of co-immunoprecipitated PML/p53 complexes in CFs treated with TGF-β1. Extracts of cells were immunoprecipitated with either anti-PML or anti-p53 antibodies and then were precipitated with either antibody (n = 5). (D) Analysis of the expression of p53 by western blot after transfection of si-PML (n = 5). (E) Analysis of the expression of p53 by western blot after transfection of PML plasmid (n = 5). (F) P53 protein levels were measured at different time points in CFs treated with cycloheximide (CHX) (10 μg/ml), CHX plus TGF-β1 by western blot analysis (n = 3). (G) PML knockdown accelerates the degradation of p53. Twenty-four hours after transfection with si-PML, CFs were incubated with CHX plus TGF-β1 for the indicated times (n = 3). (H and I) The anti-p53 antibody was used to immunoprecipitate p53-ubiquitin immunocomplexes, followed by IB analysis using the anti-Ub antibody in CFs transfected with si-PML or PML overexpression plasmid after treatment with TGF-β1 (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The membranes were incubated overnight at 4°C with the following primary antibodies: PML (1:1000; Merck Millipore, USA, #MAB3738), SUMO-1 (1:500; Abcam, USA, #ab32058), SUMO-2/3(1:500; Abcam, USA, #ab3742), p53 (1:1000; Proteintech, USA, #10442-1-AP), p-p53 (1:1000; Cell Signaling Technology, USA, #9284) and α-SMA (1:1000; Sigma, USA, #A2547), Ubiquitin (1:500; Santa Cruz Biotechnology, USA, #sc-8017), Alpha Tubulin (1:2000; Proteintech, USA, #66031l-1-lg) and GAPDH (1:10000; ABclonal, USA, #AC002).

Techniques: Binding Assay, Immunofluorescence, Western Blot, Immunoprecipitation, Expressing, Transfection, Plasmid Preparation, Knockdown, Incubation, Ubiquitin Proteomics, Over Expression

Journal: International journal of biological sciences

Article Title: Crosstalk between PML and p53 in response to TGF-β1: A new mechanism of cardiac fibroblast activation.

doi: 10.7150/ijbs.76214

Figure Lengend Snippet: Figure 6. Pharmacological inhibition of the SUMO pathway inhibits TGF-β1-induced PML/p53 interaction. (A and B) The protein levels of SUMOylated PML, SUMO-1, SUMO-2/3 and p53 in CFs treated with TGF-β1 for 6 h with or without pretreatment with GA (10 μM) for 1 h (n = 3). (C) Interaction between PML and p53 (PML/p53, red) was assessed by PLA. Scale bar, 20 μm (n = 12). ***P < 0.001.

Article Snippet: The membranes were incubated overnight at 4°C with the following primary antibodies: PML (1:1000; Merck Millipore, USA, #MAB3738), SUMO-1 (1:500; Abcam, USA, #ab32058), SUMO-2/3(1:500; Abcam, USA, #ab3742), p53 (1:1000; Proteintech, USA, #10442-1-AP), p-p53 (1:1000; Cell Signaling Technology, USA, #9284) and α-SMA (1:1000; Sigma, USA, #A2547), Ubiquitin (1:500; Santa Cruz Biotechnology, USA, #sc-8017), Alpha Tubulin (1:2000; Proteintech, USA, #66031l-1-lg) and GAPDH (1:10000; ABclonal, USA, #AC002).

Techniques: Inhibition

Journal: The Korean journal of physiology & pharmacology : official journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: Interaction of promyelocytic leukemia/p53 affects signal transducer and activator of transcription-3 activity in response to oncostatin M.

doi: 10.4196/kjpp.2020.24.3.203

Figure Lengend Snippet: Fig. 2. The status of p53 and differential protein expression level. (A) The table indicates the p53 status of cell lines used in this study. p53 status was determined through the TP53 web site (http://p53.fr). (B, up- per) NIH3T3, HEK293T, U87-MG, and U251-MG cells were either untreat- ed or treated with OSM (10 ng/ml) for 24 h, and the lysed in RIPA buffer. Whole cell lysates were analyzed by immunoblotting using anti-PML, anti-p53, anti-p-STAT-3, anti-STAT-3, anti-α-tubulin antibodies. (B, lower) The graph represents the normalized intensities of p-STAT-3 against those of STAT-3 determined from three independent experiments. WT, wild-type. **p < 0.01, ***p < 0.001 vs. untreated control.

Article Snippet: Antibody against p-STAT-3 Y705 was obtained from Cell Signaling Technology (Danvers, MA, USA) and antibodies against PML (H-238), p53 (DO-1), STAT-3 (F-2) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Expressing, Western Blot, Control

Journal: The Korean journal of physiology & pharmacology : official journal of the Korean Physiological Society and the Korean Society of Pharmacology

Article Title: Interaction of promyelocytic leukemia/p53 affects signal transducer and activator of transcription-3 activity in response to oncostatin M.

doi: 10.4196/kjpp.2020.24.3.203

Figure Lengend Snippet: Fig. 4. The effects of PML-IV and its deletion constructs on OSM-mediated STAT-3 activity. (A) Depiction of the PML-IV deletion constructs generated in this study. (B) NIH3T3, (C) U87-MG, (D) HEK293T, (E) U251-MG cells were co-transfected with the STAT-3-Luc re- porter and empty vector, PML-IV, and PML-IV deletion plasmids. At 24 h after transfection, cells were either untreated or treated with OSM (10 ng/ml) for 24 h and then assayed for luciferase activ- ity. The pCMV-β-galactosidase vector was included to normalize transfection efficiency. Data are presented as fold in- crease in relative luciferase activity (RLA) compared with RLA in the absence of OSM. PML protein expression and OSM- induced STAT-3 phosphorylation were verified for each assay by immunoblot- ting. The results are representative of three independent experiments. mOSM, murine OSM; hOSM, human OSM. *p < 0.05, ***p < 0.001 vs. untreated control; ##p < 0.01. ###p < 0.001 between cells were transfected with either PML-IV or PML-IV deletion vectors and cells were transfected with mock vector in the presence of OSM.

Article Snippet: Antibody against p-STAT-3 Y705 was obtained from Cell Signaling Technology (Danvers, MA, USA) and antibodies against PML (H-238), p53 (DO-1), STAT-3 (F-2) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Construct, Activity Assay, Generated, Transfection, Plasmid Preparation, Luciferase, Expressing, Phospho-proteomics, Western Blot, Control

Journal: Cancer Research

Article Title: Dysfunctional Microvasculature as a Consequence of Shb Gene Inactivation Causes Impaired Tumor Growth

doi: 10.1158/0008-5472.can-08-3797

Figure Lengend Snippet: Figure 5. Cytoskeleton and VEGF-dependent signaling in cultured endothelial cells isolated from Shb knockout or wild-type liver. A, the cytoskeleton of isolated endothelial cells maintained in tissue culture for 5 to 7 d was visualized by staining with rhodamine-phalloidin, showing a less regular shape with numerous extensions in Shb null cells. B, corresponding wild-type control. No clear cytoskeletal difference between wild-type or knockout cells was noted after stimulation with VEGF-A (C, Shb knockout; D, Shb wild type). Addition of VEGF to the control cells (D) produced changes that made the cells resemble the Shb null cells in the absence of VEGF-A. Horizontal scale bar is shown. Equal amounts of protein from the experimental groups (+/, 20 ng/mL VEGF-A for 2 min) were subjected to Western blot analysis for the phosphorylated proteins indicated. Shb blot shows unstimulated cells only. The blots were subjected to densitometric analysis for phosphorylated FAK, total FAK, phosphorylated p38, total p38, pMLC, phosphorylated ERK, and total ERK in three separate experiments. Quantitation of the relative phosphorylation of FAK, MLC, p38, and ERK is also given. Columns, mean; bars, SE. *, P < 0.05; **, P < 0.01 (paired Students’ t test).

Article Snippet: The samples were electrophoresed on SDS-polyacrylamide gels, protein was transferred on to Hybond-P filters (GE Healthcare), and these were then probed for pY-1175 VEGFR-2, total VEGFR-2, pY-397 FAK, total FAK, phosphorylated extracellular signal-regulated kinase (ERK), total ERK, and phosphorylated Akt, total Akt, phosphorylated myosin light chain (pMLC), pY-658 VE-cadherin, phosphorylated p38, and total p38 (all antibodies were from Cell Signaling, except for total VEGFR-2 from R&D Systems, pY-397 FAK from Biosource, pY-VE-cadherin from ProSci, and pMLC from Santa Cruz Biotechnology) before incubation with secondary antibodies and enhanced chemiluminescence.

Techniques: Cell Culture, Isolation, Knock-Out, Staining, Control, Produced, Western Blot, Quantitation Assay, Phospho-proteomics