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: Molecules and Cells

Article Title: Actin Dysfunction Induces Cell Cycle Delay at G2/M with Sustained ERK and RSK Activation in IMR-90 Normal Human Fibroblasts

doi: 10.14348/molcells.2018.2266

Figure Lengend Snippet: As denoted in , IMR-90 cells were synchronized with 2 mM double thymidine arrest, incubated with 5 μM cytochalasin D or the solvent DMSO as a control at 5.5–6 h after the second release, and collected at each indicated time point after the second release. Cell lysates were resolved by 8% SDS-PAGE and blotted. Blots were probed with (A) p-ERK1/2 and p-RSK1 (Ser 380) and re-probed with anti-ERK1/2 and anti-RSK1 to observe the total amount of each protein, (B) p-ERK1/2 and p-Cdc25C (Ser 216), and re-probed with anti-ERK1/2 and anti-Cdc25C. (A, B) Cell cycle progression at G2/M was monitored by detecting p-Cdc2 (Tyr 15) followed by re-probing with anti-Cdc2 to detect the total amount of Cdc2. (C) The same samples from (A) and (B) were blotted with p-Wee1 (Ser 642) and re-probed with anti-Wee1. Each blot was re-probed with anti-actin as a loading control.

Article Snippet: Rabbit polyclonal p-Cdc2 (Tyr 15) (ab47594, Abcam, USA), mouse monoclonal anti-cyclin A (4656, Cell Signaling Technology, USA), rabbit polyclonal p-Cdc25C (Ser 216) (9528, Cell Signaling Technology), rabbit monoclonal anti-Cdc25C (ab32444, Abcam), rabbit polyclonal anti-β-actin (4967, Cell Signaling Technology), rabbit monoclonal p-Wee1 (Ser 642) (4910, Cell Signaling Technology), rabbit polyclonal anti-Wee1 (4936, Cell Signaling Technology), rabbit monoclonal anti-FLAG (F7425, Sigma), rabbit polyclonal p-ERK1/2 (Thr 202/Tyr 204) (9101, Cell Signaling), rabbit polyclonal anti-ERK1/2 (9102, Cell Signaling), rabbit monoclonal p-RSK1 (Ser 380) (ab32203, Abcam), rabbit polyclonal anti-RSK1 (9333, Cell Signaling), rabbit monoclonal histone H3 (4499, Cell Signaling Technology), and mouse monoclonal p-H3 (Ser 10) (9706, Cell Signaling Technology) were used in this study.

Techniques: Incubation, SDS Page

Journal: Molecules and Cells

Article Title: Actin Dysfunction Induces Cell Cycle Delay at G2/M with Sustained ERK and RSK Activation in IMR-90 Normal Human Fibroblasts

doi: 10.14348/molcells.2018.2266

Figure Lengend Snippet: (A) The layout shows how the experiment was performed. IMR-90 cells were synchronized with 2 mM thymidine and transfected with DN-RSK or empty vector as described in the Materials and Methods. Cells were treated with 5 μM CD or only DMSO as a control at 5.5–6 h after the second release and collected at each indicated time point after the second release. Each cell lysate was resolved by 8% SDS-PAGE and blotted with (B) anti-FLAG and anti-actin, (C) p-ERK1/2 and p-Cdc2 (Tyr 15), (D) p-Cdc25C (Ser 216), and p-Wee1 (Ser 642). Each membrane was reprobed with anti-actin as a loading control.

Article Snippet: Rabbit polyclonal p-Cdc2 (Tyr 15) (ab47594, Abcam, USA), mouse monoclonal anti-cyclin A (4656, Cell Signaling Technology, USA), rabbit polyclonal p-Cdc25C (Ser 216) (9528, Cell Signaling Technology), rabbit monoclonal anti-Cdc25C (ab32444, Abcam), rabbit polyclonal anti-β-actin (4967, Cell Signaling Technology), rabbit monoclonal p-Wee1 (Ser 642) (4910, Cell Signaling Technology), rabbit polyclonal anti-Wee1 (4936, Cell Signaling Technology), rabbit monoclonal anti-FLAG (F7425, Sigma), rabbit polyclonal p-ERK1/2 (Thr 202/Tyr 204) (9101, Cell Signaling), rabbit polyclonal anti-ERK1/2 (9102, Cell Signaling), rabbit monoclonal p-RSK1 (Ser 380) (ab32203, Abcam), rabbit polyclonal anti-RSK1 (9333, Cell Signaling), rabbit monoclonal histone H3 (4499, Cell Signaling Technology), and mouse monoclonal p-H3 (Ser 10) (9706, Cell Signaling Technology) were used in this study.

Techniques: Transfection, Plasmid Preparation, SDS Page