Journal:

Article Title: Sphingosine 1-phosphate promotes endothelial cell barrier integrity by Edg-dependent cytoskeletal rearrangement

doi:

Figure Lengend Snippet: Role of Rac GTPase in Sph-1-P–mediated endothelial actin rearrangement and barrier regulation. (a) Bovine pulmonary artery endothelium were incubated with vehicle (v) Sph-1-P (1 μM), for indicated periods of time, or pretreated with PTX (1 μg/ml, 2 hours) and subsequently incubated with Sph-1-P for 1 minute. Cells were lysed, centrifuged, and supernatants were collected. The activated GTP-bound Rac was precipitated by agarose-conjugated human PAK-1 p21-binding domain and immunoblotted by anti-Rac mAb as described in Methods. Total Rac content was detected using cell lysates. These results indicate rapid Sph-1-P–induced Rac activation, which was completely abolished by PTX. (b–g) Bovine endothelium was transfected with either an empty vector (b and e) or a constitutively active HA-tagged Rac construct (Rac V12) (c, d, f, and g) as described in Methods. Shown are subsequent immunofluorescence images (×100) of endothelial cells stained with either Texas red phalloidin for F-actin (b–d) or anti-HA tag Ab for identification of transfected cells (e–g). b and e, c and f, d and g represent matched images. Overexpression of constitutively active Rac V12, but not control vector, significantly enhances polymerized actin staining (arrows) within the cortical ring with the degree of enhancement dependent upon the level of Rac V-12 overexpression.

Article Snippet: After a brief centrifugation to remove the cell debris, 300 μl of supernatants were incubated with human p21-binding PAK-1 domain (residues 67-150), conjugated with agarose (10 μg, 30 minutes; Upstate Biotechnology Inc.).

Techniques: Incubation, Binding Assay, Activation Assay, Transfection, Plasmid Preparation, Construct, Immunofluorescence, Staining, Over Expression

Journal: PLoS ONE

Article Title: Pax3 Stimulates p53 Ubiquitination and Degradation Independent of Transcription

doi: 10.1371/journal.pone.0029379

Figure Lengend Snippet: (A) Pulse labeling with 35 S-met to determine the rate of p53 synthesis in stage1 and stage 3 ESC. Quantitation of 35 S-p53 is described in the supporting online material. (B) Pulse-chase labeling to determine the t 12 of p53 in stage 1 and stage 3 ESC. (C) Quantitation of ubiquitinated p53/total p53 in stage 1 and stage 3 ESC following immunoprecipitation of p53 and immunoblotting using anti-ubiquitin or anti-p53 antibodies. *p<0.05 vs. stage 1. (D) Whole cell extracts of stage 1 or stage 3 ESC were immunoprecipitated using antibodies against p53, Pax3 or Mdm2, and then immunoblotted using antibodies against p53, Pax3, and Mdm2. (E) Schematic diagram of full-length w.t. Pax3, Pax3 structural domains, and a Splotch Pax3 (Sp) protein product that were expressed as GST and FLAG fusion proteins. N-term, amino-terminus of Pax3 through the homeodomain; C-term, carboxy-terminus distal to the homeodomain (including the trans activation domain); DBD, DNA-binding domain (PD through HD); ID, the trans-activation inhibitory domain (amino-terminal to the PD); PD, paired domain; OCT, octapeptide (carboxy-terminal of the PD to amino-terminal of the HD); and HD, homeodomain. The Splotch cDNA deletes exon 4 and lacks coding sequence for part of the PD and the OCT but retains the HD. Numbers refer to amino acid positions of w.t. Pax3. (F) Immunoblot using antibodies against p53 (upper panel), Mdm2 (middle panel), or GST following incubation of whole cell lysates from stage 1 ESC with GST-Pax3 fusion proteins linked to glutathione-sepharose beads. (G) Immunoblot using antibodies against p53 (upper panel), Mdm2 (middle panel), or FLAG following incubation of whole cell lysates from stage 1 ESC that had been transiently transfected with plasmids encoding FLAG-tagged Pax3 fusion proteins with antibody against FLAG linked to M2 beads.

Article Snippet: The reaction mixture (20 μl) contained 10 ng GST-p53 (murine), 24 ng E1 (Boston Biochem), 20 ng GST-UbcH5C (Boston Biochem), 150 ng GST-Mdm2 (murine), 10 μg His-ubiquitin (Boston Biochem), plus GST fusion proteins containing full length Pax3 or Pax3 domains.

Techniques: Labeling, Quantitation Assay, Pulse Chase, Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Activation Assay, Binding Assay, Sequencing, Incubation, Transfection

Journal: PLoS ONE

Article Title: Pax3 Stimulates p53 Ubiquitination and Degradation Independent of Transcription

doi: 10.1371/journal.pone.0029379

Figure Lengend Snippet: (A) In vitro ubiquitination reactions of GST-p53 with GST-Mdm2 (150 ng), and 0–1600 ng GST-Pax3. Ubiquitination was assayed by immunoblot using 53 antibodies. The position of GST-p53 is indicated by a heavy arrow, and the positions of ubiquitinated p53 are indicated by narrow arrows. (B) In vitro ubiquitination reactions as in (A) except that GST without any Pax3 coding sequences was used. (C) In vitro ubiquitination reactions performed with only GST-Mdm2 or 400 ng GST-Pax3. (D) In vitro ubiquitination reactions of GST-p53 with GST-Mdm2 and 400 ng GST fusion proteins of w.t. Pax3, Pax3 structural domains, or Splotch Pax3. (The GST-p53 and GST-Mdm2 vectors used here encoded murine p53 and Mdm2, although similar results were obtained using human p53 and Mdm2 fusion proteins (data not shown).)

Article Snippet: The reaction mixture (20 μl) contained 10 ng GST-p53 (murine), 24 ng E1 (Boston Biochem), 20 ng GST-UbcH5C (Boston Biochem), 150 ng GST-Mdm2 (murine), 10 μg His-ubiquitin (Boston Biochem), plus GST fusion proteins containing full length Pax3 or Pax3 domains.

Techniques: In Vitro, Ubiquitin Proteomics, Western Blot