Journal: PLoS ONE

Article Title: c-Rel Deficiency Increases Caspase-4 Expression and Leads to ER Stress and Necrosis in EBV-Transformed Cells

doi: 10.1371/journal.pone.0025467

Figure Lengend Snippet: ( A ) Pt1- (dashed line) and control D11-LCL tet cells (solid line) grown in the absence of Tc for 5 days were plated at 1×10 6 cells/mL in Tc media and cultured in parallel for 6 days. Cells were withdrawn at days 1, 3 and 6 cells and counted using Trypan-Blue exclusion to determine the total number of viable cells. ( B ) 1×10 6 Pt1- and control D11- and C2-LCL tet cells were stained with CFSE and cultured in Tc media (pink line), absence of Tc (for 5 days prior to CFSE staining) (grey peak) or absence of Tc plus sCD40L (500 ng/mL) (green line) for 6 days. At day 4 (upper panel) and day 6 (lower panel) cells were analyzed for proliferation by flow cytometry. ( C ) Pt1- and control D11-LCL tet cells (2×10 5 ) grown in Tc media were analyzed for the expression of phosphorylated-CDC2 by intra-cellular staining. Numbers shown are the mean fluorescence intensity (MFI) of cells expressing high levels of phosphorylated CDC2. Graph is representative of three independent experiments.

Article Snippet: Anti-S6 mAb, rabbit polyclonal anti-caspase 4 and rabbit polyclonal anti-phospho-CDC2 were purchased from Cell Signaling.

Techniques: Cell Culture, Staining, Flow Cytometry, Expressing, Fluorescence

Journal: Retrovirology

Article Title: Human T-lymphotropic virus type-1 p30 alters cell cycle G2 regulation of T lymphocytes to enhance cell survival

doi: 10.1186/1742-4690-4-49

Figure Lengend Snippet: p30 enhances phosphorylation of Cdc25C at S-216 . A) 40 ug of nuclear (N) or cytosolic (C) fraction prepared from either p30 expressing or Mock Jurkat T-cells were loaded on 10% SDS gel and western blot analysis was performed with anti-HA to confirm p30 expression and Histone H1 western to confirm nuclear and cytosolic fractionation. B) Western blot of nuclear (N) and cytosolic (C) extracts prepared from p30 or mock Jurkat T-cells probed with anti-Cdc2 and phosphospecific Cdc2 antibody. C) Western blot of nuclear (N) and cytosolic (C) extracts prepared from p30 or mock Jurkat T-cells, probed with anti-Cdc25C or phosphospecific anti-Cdc25C. D) Western Blot analysis of nuclear or cytosolic extracts from p30 or mock Jurkat T-cells, probed with anti-Chk1 or monoclonal phosphospecific Chk1 (S-345).

Article Snippet: Immunodetection was performed using the following antibodies: mouse anti-HA monoclonal antibody clone 16B-12 (1:1000, Covance Research Products, Princeton, NJ), mouse monoclonal anti-PARP clone C-2-10 (1:1000, Oncogene Research Products, Boston, MA). mouse monoclonal anti-β-actin clone AC-74 (1:4000, Sigma), rabbit polyclonal anti-Cdc2 # 9112 (1:1000, Cell Signaling, Beverly, MA), rabbit polyclonal anti-PLK1pT210 # 600-401-466 (1: 1000, Rockland, Gilbertsville, PA), polyclonal rabbit anti-Cdc2 T-161 #9114 (1:1000, Cell Signaling), polyclonal rabbit anti Cdc2 Y-15 #9111(1:1000, Cell Signaling), polyclonal rabbit anti-Cdc25C #9522 (1:1000, Cell Signaling), polyclonal rabbit anti-Cdc25C S-216 # 9528(1:1000, Cell Signaling), rabbit polyclonal anti-Cdc25C pS-198 # 9529 (1: 1000, Cell Signaling), polyclonal rabbit anti-Chk1 #2345 (1:1000, Cell Signaling), polyclonal rabbit anti-Chk1 S-345 #2341 (1: 1000, Cell Signaling), rabbit polyclonal anti-PLK1 (1: 1000) (Abcam, Cambridge, MA), mouse monoclonal anti-Histone H1, clone AE-4 (1: 1000, Upstate).

Techniques: Expressing, SDS-Gel, Western Blot, Fractionation

Journal: Retrovirology

Article Title: Human T-lymphotropic virus type-1 p30 alters cell cycle G2 regulation of T lymphocytes to enhance cell survival

doi: 10.1186/1742-4690-4-49

Figure Lengend Snippet: p30 enhances phosphorylation of Cdc25C at S-216 . A) 40 ug of nuclear (N) or cytosolic (C) fraction prepared from either p30 expressing or Mock Jurkat T-cells were loaded on 10% SDS gel and western blot analysis was performed with anti-HA to confirm p30 expression and Histone H1 western to confirm nuclear and cytosolic fractionation. B) Western blot of nuclear (N) and cytosolic (C) extracts prepared from p30 or mock Jurkat T-cells probed with anti-Cdc2 and phosphospecific Cdc2 antibody. C) Western blot of nuclear (N) and cytosolic (C) extracts prepared from p30 or mock Jurkat T-cells, probed with anti-Cdc25C or phosphospecific anti-Cdc25C. D) Western Blot analysis of nuclear or cytosolic extracts from p30 or mock Jurkat T-cells, probed with anti-Chk1 or monoclonal phosphospecific Chk1 (S-345).

Article Snippet: Immunodetection was performed using the following antibodies: mouse anti-HA monoclonal antibody clone 16B-12 (1:1000, Covance Research Products, Princeton, NJ), mouse monoclonal anti-PARP clone C-2-10 (1:1000, Oncogene Research Products, Boston, MA). mouse monoclonal anti-β-actin clone AC-74 (1:4000, Sigma), rabbit polyclonal anti-Cdc2 # 9112 (1:1000, Cell Signaling, Beverly, MA), rabbit polyclonal anti-PLK1pT210 # 600-401-466 (1: 1000, Rockland, Gilbertsville, PA), polyclonal rabbit anti-Cdc2 T-161 #9114 (1:1000, Cell Signaling), polyclonal rabbit anti Cdc2 Y-15 #9111(1:1000, Cell Signaling), polyclonal rabbit anti-Cdc25C #9522 (1:1000, Cell Signaling), polyclonal rabbit anti-Cdc25C S-216 # 9528(1:1000, Cell Signaling), rabbit polyclonal anti-Cdc25C pS-198 # 9529 (1: 1000, Cell Signaling), polyclonal rabbit anti-Chk1 #2345 (1:1000, Cell Signaling), polyclonal rabbit anti-Chk1 S-345 #2341 (1: 1000, Cell Signaling), rabbit polyclonal anti-PLK1 (1: 1000) (Abcam, Cambridge, MA), mouse monoclonal anti-Histone H1, clone AE-4 (1: 1000, Upstate).

Techniques: Expressing, SDS-Gel, Western Blot, Fractionation

Journal: Retrovirology

Article Title: Human T-lymphotropic virus type-1 p30 alters cell cycle G2 regulation of T lymphocytes to enhance cell survival

doi: 10.1186/1742-4690-4-49

Figure Lengend Snippet: p30 enhances phosphorylation of Cdc25C at S-216 . A) 40 ug of nuclear (N) or cytosolic (C) fraction prepared from either p30 expressing or Mock Jurkat T-cells were loaded on 10% SDS gel and western blot analysis was performed with anti-HA to confirm p30 expression and Histone H1 western to confirm nuclear and cytosolic fractionation. B) Western blot of nuclear (N) and cytosolic (C) extracts prepared from p30 or mock Jurkat T-cells probed with anti-Cdc2 and phosphospecific Cdc2 antibody. C) Western blot of nuclear (N) and cytosolic (C) extracts prepared from p30 or mock Jurkat T-cells, probed with anti-Cdc25C or phosphospecific anti-Cdc25C. D) Western Blot analysis of nuclear or cytosolic extracts from p30 or mock Jurkat T-cells, probed with anti-Chk1 or monoclonal phosphospecific Chk1 (S-345).

Article Snippet: Immunodetection was performed using the following antibodies: mouse anti-HA monoclonal antibody clone 16B-12 (1:1000, Covance Research Products, Princeton, NJ), mouse monoclonal anti-PARP clone C-2-10 (1:1000, Oncogene Research Products, Boston, MA). mouse monoclonal anti-β-actin clone AC-74 (1:4000, Sigma), rabbit polyclonal anti-Cdc2 # 9112 (1:1000, Cell Signaling, Beverly, MA), rabbit polyclonal anti-PLK1pT210 # 600-401-466 (1: 1000, Rockland, Gilbertsville, PA), polyclonal rabbit anti-Cdc2 T-161 #9114 (1:1000, Cell Signaling), polyclonal rabbit anti Cdc2 Y-15 #9111(1:1000, Cell Signaling), polyclonal rabbit anti-Cdc25C #9522 (1:1000, Cell Signaling), polyclonal rabbit anti-Cdc25C S-216 # 9528(1:1000, Cell Signaling), rabbit polyclonal anti-Cdc25C pS-198 # 9529 (1: 1000, Cell Signaling), polyclonal rabbit anti-Chk1 #2345 (1:1000, Cell Signaling), polyclonal rabbit anti-Chk1 S-345 #2341 (1: 1000, Cell Signaling), rabbit polyclonal anti-PLK1 (1: 1000) (Abcam, Cambridge, MA), mouse monoclonal anti-Histone H1, clone AE-4 (1: 1000, Upstate).

Techniques: Expressing, SDS-Gel, Western Blot, Fractionation

Journal: Retrovirology

Article Title: Human T-lymphotropic virus type-1 p30 alters cell cycle G2 regulation of T lymphocytes to enhance cell survival

doi: 10.1186/1742-4690-4-49

Figure Lengend Snippet: p30 enhances phosphorylation of Cdc25C at S-216 . A) 40 ug of nuclear (N) or cytosolic (C) fraction prepared from either p30 expressing or Mock Jurkat T-cells were loaded on 10% SDS gel and western blot analysis was performed with anti-HA to confirm p30 expression and Histone H1 western to confirm nuclear and cytosolic fractionation. B) Western blot of nuclear (N) and cytosolic (C) extracts prepared from p30 or mock Jurkat T-cells probed with anti-Cdc2 and phosphospecific Cdc2 antibody. C) Western blot of nuclear (N) and cytosolic (C) extracts prepared from p30 or mock Jurkat T-cells, probed with anti-Cdc25C or phosphospecific anti-Cdc25C. D) Western Blot analysis of nuclear or cytosolic extracts from p30 or mock Jurkat T-cells, probed with anti-Chk1 or monoclonal phosphospecific Chk1 (S-345).

Article Snippet: Immunodetection was performed using the following antibodies: mouse anti-HA monoclonal antibody clone 16B-12 (1:1000, Covance Research Products, Princeton, NJ), mouse monoclonal anti-PARP clone C-2-10 (1:1000, Oncogene Research Products, Boston, MA). mouse monoclonal anti-β-actin clone AC-74 (1:4000, Sigma), rabbit polyclonal anti-Cdc2 # 9112 (1:1000, Cell Signaling, Beverly, MA), rabbit polyclonal anti-PLK1pT210 # 600-401-466 (1: 1000, Rockland, Gilbertsville, PA), polyclonal rabbit anti-Cdc2 T-161 #9114 (1:1000, Cell Signaling), polyclonal rabbit anti Cdc2 Y-15 #9111(1:1000, Cell Signaling), polyclonal rabbit anti-Cdc25C #9522 (1:1000, Cell Signaling), polyclonal rabbit anti-Cdc25C S-216 # 9528(1:1000, Cell Signaling), rabbit polyclonal anti-Cdc25C pS-198 # 9529 (1: 1000, Cell Signaling), polyclonal rabbit anti-Chk1 #2345 (1:1000, Cell Signaling), polyclonal rabbit anti-Chk1 S-345 #2341 (1: 1000, Cell Signaling), rabbit polyclonal anti-PLK1 (1: 1000) (Abcam, Cambridge, MA), mouse monoclonal anti-Histone H1, clone AE-4 (1: 1000, Upstate).

Techniques: Expressing, SDS-Gel, Western Blot, Fractionation

Journal: Retrovirology

Article Title: Human T-lymphotropic virus type-1 p30 alters cell cycle G2 regulation of T lymphocytes to enhance cell survival

doi: 10.1186/1742-4690-4-49

Figure Lengend Snippet: p30 enhances phosphorylation of Cdc25C at S-216 . A) 40 ug of nuclear (N) or cytosolic (C) fraction prepared from either p30 expressing or Mock Jurkat T-cells were loaded on 10% SDS gel and western blot analysis was performed with anti-HA to confirm p30 expression and Histone H1 western to confirm nuclear and cytosolic fractionation. B) Western blot of nuclear (N) and cytosolic (C) extracts prepared from p30 or mock Jurkat T-cells probed with anti-Cdc2 and phosphospecific Cdc2 antibody. C) Western blot of nuclear (N) and cytosolic (C) extracts prepared from p30 or mock Jurkat T-cells, probed with anti-Cdc25C or phosphospecific anti-Cdc25C. D) Western Blot analysis of nuclear or cytosolic extracts from p30 or mock Jurkat T-cells, probed with anti-Chk1 or monoclonal phosphospecific Chk1 (S-345).

Article Snippet: Immunodetection was performed using the following antibodies: mouse anti-HA monoclonal antibody clone 16B-12 (1:1000, Covance Research Products, Princeton, NJ), mouse monoclonal anti-PARP clone C-2-10 (1:1000, Oncogene Research Products, Boston, MA). mouse monoclonal anti-β-actin clone AC-74 (1:4000, Sigma), rabbit polyclonal anti-Cdc2 # 9112 (1:1000, Cell Signaling, Beverly, MA), rabbit polyclonal anti-PLK1pT210 # 600-401-466 (1: 1000, Rockland, Gilbertsville, PA), polyclonal rabbit anti-Cdc2 T-161 #9114 (1:1000, Cell Signaling), polyclonal rabbit anti Cdc2 Y-15 #9111(1:1000, Cell Signaling), polyclonal rabbit anti-Cdc25C #9522 (1:1000, Cell Signaling), polyclonal rabbit anti-Cdc25C S-216 # 9528(1:1000, Cell Signaling), rabbit polyclonal anti-Cdc25C pS-198 # 9529 (1: 1000, Cell Signaling), polyclonal rabbit anti-Chk1 #2345 (1:1000, Cell Signaling), polyclonal rabbit anti-Chk1 S-345 #2341 (1: 1000, Cell Signaling), rabbit polyclonal anti-PLK1 (1: 1000) (Abcam, Cambridge, MA), mouse monoclonal anti-Histone H1, clone AE-4 (1: 1000, Upstate).

Techniques: Expressing, SDS-Gel, Western Blot, Fractionation

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Leaf Extracts of Calocedrus formosana (Florin) Induce G2/M Cell Cycle Arrest and Apoptosis in Human Bladder Cancer Cells

doi: 10.1155/2011/380923

Figure Lengend Snippet: Western blot analysis of cell cycle regulatory protein levels and apoptosis proteins in TCCSUP cells treated with Florin extracts. (a) Cdc25C, Cdc-2, pCdc-2, cyclin-B1, and Bcl-2 proteins were analyzed after cells were exposed to Florin extracts. Cells were treated with 25 μ g/mL of Florin extracts for 6, 12, 24, and 36 h. Proteins (50 μ g) from each sample was resolved on 12% SDS-PAGE and Western blot was performed. GAPDH was used as a control. The thick and thin bars indicate the locations of the fast (dephosphorylated) and slow (phosphorylated) migrating forms of cyclin B1, P-Cdc2, or Cdc2, respectively. (b) Expression of Bcl-2 and Bax proteins in TCCSUP cells was analyzed. Cells were treated with 6, 12, and 25 μ g/mL of Florin extracts for 24 h. Proteins (50 μ g) from each sample were resolved on 12% SDS-PAGE and Western blot was performed. GAPDH was used as a control. The two slow migrating forms of Bcl2 may be the hyperphosphorylated forms of Bcl2.

Article Snippet: Cell lysates with equal amounts of proteins, which were measured using a BCA Protein Reagent Kit (Pierce, Rockford, IL, USA), were analyzed by Western blot, using a rabbit polyclonal antibody to cdc2 phosphorylated at Tyr15 (p-Cdc2) (1 : 1000; R&D, Minneapolis, MN, USA), a rabbit polyclonal cdc2 antibody (1 : 1000; Cell Signaling Technology, St. Louis, MO, USA), a mouse monoclonal antibody to cyclin B1 (sc-254; 1 : 200) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), a mouse monoclonal antibody to poly (ADP-ribose) polymerase (PARP) (0.5 μ g/mL; from BD Biosciences, Franklin Lakes, NJ, USA), a mouse monoclonal antibody to Bcl-2, a rabbit polyclonal antibody to Bax (1 : 200; both from DAKO, Taipei, Taiwan), an anti- β -tubulin mouse monoclonal antibody (1 : 20000; Epitomics, Burlingame, California), or a mouse monoclonal antibody against human GAPDH (1 : 1000; Santa Cruz, CA, USA).

Techniques: Western Blot, SDS Page, Expressing

Journal: PLoS ONE

Article Title: 14-3-3 σ Expression Effects G2/M Response to Oxygen and Correlates with Ovarian Cancer Metastasis

doi: 10.1371/journal.pone.0015864

Figure Lengend Snippet: Protein lysates prepared from the ovarian cancer cell lines maintained in growth medium consisting of increasing concentrations of serum and 21% or 3% O 2 were analyzed by Western blot. (A) Compared to O 2 sensitive cell lines, decreased expression of the core components involved in G2/M cell cycle progression CDC2/cyclin B1 complex and its activator CDC25c is observed in the O 2 insensitive cell lines (indicated by asterisk and italics), while the expression of 14-3-3 σ, a protein that inhibits CDC2 is elevated in the O 2 insensitive cell lines. (B) Phosphorylation of RB and Wee1 were monitored as an indicator for CDC2 function because both RB and Wee1 are known targets for phosphorylation by CDC2. Equal loading of protein extracts were monitored by probing the stripped Western blots with the primary antibody for β-actin.

Article Snippet: The primary antibodies used were mouse monoclonal 14-3-3 σ at 1.0 µg/mL (Upstate) and rabbit polyclonal total CDC2 at 1∶1000 (Cell Signaling).

Techniques: Western Blot, Expressing

Journal: PLoS ONE

Article Title: 14-3-3 σ Expression Effects G2/M Response to Oxygen and Correlates with Ovarian Cancer Metastasis

doi: 10.1371/journal.pone.0015864

Figure Lengend Snippet: (A) Cellular localization by immunofluoresence shows that 14-3-3 σ (Green) is located in the cytoplasm and CDC2 (Red) is present in the nucleus (Blue). Compared to O 2 sensitive A2780 cells, the level of 14-3-3 σ is higher and CDC2 is low in the O 2 insensitive HeyA8 cells. In the O 2 sensitive A2780, 14-3-3 σ is localized both in the nucleus and cytoplasm at 21% O 2 . (A dotted yellow line, outlines a representative nuclei to indicate relative localization of 14-3-3 σ and CDC2 in these cells). (B) Western blot analysis of nuclear and cytoplasmic fractions show low levels of 14-3-3 σ in the nucleus compared to cytoplasm, with increased amounts of 14-3-3 σ being present in the cytoplasm of the O 2 insensitive HeyA8 cells. The level of CDC2 is higher both in the nucleus and cytoplasm of the O 2 sensitive A2780, but present in lower amount only in the nucleus of O 2 insensitive HeyA8 cells. Histone H1 and β−actin were used as loading controls for nuclear and cytoplasmic fractions, respectively. (C) Mitotic cells were determined by counting the cells that stained positively for a mitosis specific marker, Phospho-Histone H3 from the total cell population. Mitotic fractions present at 3% or 21% O 2 were counted in both A2780 and HeyA8 and represented as bar graph. A significant increase in mitotic index (p>0.001, indicated by asterisk) was observed in the O 2 sensitive A2780 at 3% O 2 , but not in the O 2 insensitive HeyA8 cells. (D) Over-expression of 14-3-3 σ in the O 2 sensitive A2780 (Western Blot) results in loss of O 2 sensitivity (Bar graph). For the cells transfected with empty vector (mock transfection) or 14-3-3 σ over-expression construct, the percent of cell proliferation was compared with proliferation of mock transfected cells grown under standard tissue culture conditions consisting of 21% O 2 (ambient), and (E) in the converse experiment performed with O 2 insensitive HeyA8, reducing the levels of 14-3-3 σ by siRNA (Western blot) results in restoration of O 2 sensitivity (Bar graph). For the cells transfected with scrambled siRNA (mock transfection) or siRNA against 14-3-3 σ, the percent of cell proliferation was compared with proliferation of mock transfected cells grown under standard tissue culture conditions consisting of 21% O 2.

Article Snippet: The primary antibodies used were mouse monoclonal 14-3-3 σ at 1.0 µg/mL (Upstate) and rabbit polyclonal total CDC2 at 1∶1000 (Cell Signaling).

Techniques: Western Blot, Staining, Marker, Over Expression, Transfection, Plasmid Preparation, Construct

Journal: PLoS ONE

Article Title: 14-3-3 σ Expression Effects G2/M Response to Oxygen and Correlates with Ovarian Cancer Metastasis

doi: 10.1371/journal.pone.0015864

Figure Lengend Snippet: (A) Hierarchical clustering of normalized RPPA data over Phospho-RB (Ser 807/811), 14-3-3 σ, CDC2 and p53 across 57 ovarian cancer cell lines. (B) Hierarchical clustering of normalized RPPA data over Phospho-RB (Ser 807/811), 14-3-3 σ, CDC2 and p53 across 205 ovarian tumors. The color codes for overall survival represents overall survival >24 months (blue) and overall survival <24 months (pink). The color codes for tumor stage represent stage I (red), stage II (green), stage III (light-blue) and stage IV (dark-blue). (C). Kaplan-Meier survival curve for the RPPA results comparing the group of ovarian tumors with high Phospho-RB and high 14-3-3 σ or CDC2 (blue line) with other expression profiles (red line).

Article Snippet: The primary antibodies used were mouse monoclonal 14-3-3 σ at 1.0 µg/mL (Upstate) and rabbit polyclonal total CDC2 at 1∶1000 (Cell Signaling).

Techniques: Expressing