Journal: Free Radical Biology and Medicine

Article Title: CELL CYCLE ARREST BY MONOCHLORAMINE THROUGH THE OXIDATION OF RETINOBLASTOMA PROTEIN

doi: 10.1016/j.freeadbiomed.2003.10.013

Figure Lengend Snippet: Fig. 2. Phosphorylation status of pRB after NH2Cl treatment. Jurkat cells were treated with 70 AM NH2Cl for 10 min at 37jC. Then the medium was replaced with fresh medium, and incubation proceeded for the indicated times. The phosphorylation status of pRB was studied by the Western blotting method. (A) Immunostaining with anti-pRB antibody. Hypo-phosphorylated pRB migrates slightly faster than hyperphos- phorylated pRB on SDS–PAGE. (B) Immunostaining with phospho- specific antibodies that react only when pRB is phosphorylated at the specific serine residue. Optical densities were measured with Scion image software and are expressed as percentages of ‘‘time 0’’ samples.

Article Snippet: Antibodies specific to the phosphorylated pRB (Ser780, Ser795 and Ser807/811) were from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Phospho-proteomics, Incubation, Western Blot, Immunostaining, SDS Page, Residue, Software

Journal: Free Radical Biology and Medicine

Article Title: CELL CYCLE ARREST BY MONOCHLORAMINE THROUGH THE OXIDATION OF RETINOBLASTOMA PROTEIN

doi: 10.1016/j.freeadbiomed.2003.10.013

Figure Lengend Snippet: Fig. 5. Effects of NH2Cl on protein phosphatase. (A) GST-pRB was pre-phosphorylated as described under Materials and Methods, then adsorbed to glutathione–agarose beads and used for phosphatase substrate. The beads were incubated with the soluble fraction of the control and NH2Cl-treated cell lysate for 90 min at 37jC, and the products were analyzed by Western blotting using phospho-specific antibody. The faint band indicates high phosphatase activity. The lower panel shows the equal load of the substrate protein. (B) Immunoblot of protein phosphatase 1. Jurkat cells were treated with NH2Cl and the cell lysate was prepared as described in the legend to Fig. 2. Expression of protein phosphatase 1, a major pRB phosphatase, was studied by SDS– PAGE and Western blotting.

Article Snippet: Antibodies specific to the phosphorylated pRB (Ser780, Ser795 and Ser807/811) were from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Incubation, Control, Western Blot, Activity Assay, Expressing, SDS Page

Journal: PLoS ONE

Article Title: An Opposite Effect of the CDK Inhibitor, p18 INK4c on Embryonic Stem Cells Compared with Tumor and Adult Stem Cells

doi: 10.1371/journal.pone.0045212

Figure Lengend Snippet: (A, B and C) Real-time RT-PCR analysis of p21 , p27 , and CDK2 mRNA levels. All values were normalized to β-actin. Values are expressed as the mean ± SD. (D) Western blotting performed to detect protein levels of p18, CDK2, CDK4, CDK6, pRb-Ser795, pRb-Ser811, and total Rb. Detection of β-actin was used as a loading control. (E) IP assays of p18, p21 and p27 were performed using cell lysate (100 µg total protein) from stably transduced p18, or WT ES cells. Immunocomplexes obtained were then immunoblotted with anti-CDK4 and anti-CDK2 antibodies. (F) Total protein extracts were obtained from stably transduced p18, or WT ES cells and immunoblotted with anti-p18, anti-p21, anti-p27, and anti-cdk2 and anti-cdk4 antibodies. β-actin was used as a loading control. (G) A model for a proposed mechanism by which p18 enhances the self-renewal of ES cells, while inhibiting their differentiation potential. Briefly, ectopic expression of p18 promotes overexpression of CDK4, which in turn enhances the association of p21 and p27 with CDK4, and ultimately upregulates CDK2 activities. As a result, the cell cycle is accelerated and the self-renewal is enhanced, whereas the differentiation process is slowed. In A to F, data represent three independent experiments with similar results.

Article Snippet: The antibodies against CDK4 (C-22), p21 (F-5), CDK2 (M2), CDK6 (H-230), p27 (C-19), Rb (M-153), and Polyclonal antibodies against pRb (Ser 807/811)-R) and pRb (Ser 795)-R) were from Santa Cruz Biotechnology, Inc., (Santa Cruz, CA).

Techniques: Quantitative RT-PCR, Western Blot, Control, Stable Transfection, Expressing, Over Expression

Journal: PLoS ONE

Article Title: An Opposite Effect of the CDK Inhibitor, p18 INK4c on Embryonic Stem Cells Compared with Tumor and Adult Stem Cells

doi: 10.1371/journal.pone.0045212

Figure Lengend Snippet: Sequences of the Primers used for Real-time RT-PCR assays.

Article Snippet: The antibodies against CDK4 (C-22), p21 (F-5), CDK2 (M2), CDK6 (H-230), p27 (C-19), Rb (M-153), and Polyclonal antibodies against pRb (Ser 807/811)-R) and pRb (Ser 795)-R) were from Santa Cruz Biotechnology, Inc., (Santa Cruz, CA).

Techniques: Quantitative RT-PCR