Journal: Journal of Biological Chemistry

Article Title: PTEN Deletion Leads to Up-regulation of a Secreted Growth Factor Pleiotrophin

doi: 10.1074/jbc.m512509200

Figure Lengend Snippet: FIGURE3.PtnexpressionisregulatedbyPI3K/AKTpathway.A,MEFcellsweretreated with phosphoinositide 3-kinase inhibitor LY294002 (10 M, LY10; and 30 M, LY30) or mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 (50 M) for 24 h, then subjected to Northern hybridization. B, PTEN suppresses the expression of Ptn. 3T3 cells were transfected with the indicated constructs, and subjected to Northern analysis 24 h after transfection. Pten WT, construct containing wild-type Pten cDNA; Pten CS, construct containing Pten cDNA with the C124S point mutation that lacks phosphatase activity. Empty pIRES2-EGFP vector was transfected as control. C, overexpression of wild-type or the activated form of AKT results in up-regulation of Ptn. 3T3 cells were transfected with the indicated constructs, and subjected to Northern analysis 24 h after transfection. Akt1 WT, construct containing wild-type Akt1 cDNA; Akt1 DN, construct containing dominant negative Akt1 cDNA; Akt1 AC, construct containing activated form of Akt1 cDNA. Empty pIRES2-EGFP vector was transfected as control. The amount of ribosomal RNAs was used as loading control in each panel. Signal intensities of the Northern blot were quantified by Quantity One (Bio-Rad), normalized by the intensities of 28 S ribosomal RNA, and graphed at the right.

Article Snippet: Western blots were then probed with the antibodies against PTN (R&D Systems), and AKT, phospho-AKT (Thr-308), phospho-GSK-3 and -actin, which were all purchased fromCell Signaling Technology.

Techniques: Northern Blot, Hybridization, Expressing, Transfection, Construct, Mutagenesis, Activity Assay, Plasmid Preparation, Control, Over Expression, Dominant Negative Mutation

Journal: Cell Death & Disease

Article Title: Serpina3n attenuates granzyme B-mediated decorin cleavage and rupture in a murine model of aortic aneurysm

doi: 10.1038/cddis.2011.88

Figure Lengend Snippet: GZMB cleavage of decorin is prevented by pre-incubation with SA3N in vitro . Purified GZMB was pre-incubated with or without SA3N for 25 min at RT before incubation with recombinant human decorin for 24 h at RT. Proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane and visualized by western blotting for decorin ( a ) and Ponceau stain ( b ). In a , full-length decorin is indicated by an asterisk ( * ) and is visible at ∼65 kDa in lane 1. Decorin is severely reduced following incubation with GZMB in lane 2. In lane 3, decorin loss is not observed following pre-incubation of GZMB with SA3N. In b , SA3N and SA3N–GZMB complex at approximately 47 and 70 kDa, respectively (indicated by ^) are visible in lanes 2, 3 and 6. Full-length decorin is indicated by the asterisk ( * ) at approximately 65 kDa in lanes 4, 5 and 6. Decorin cleavage fragments in lane 5 are marked by arrows on the right side of the membrane

Article Snippet: The membrane was probed using a mouse anti-human decorin antibody (1 : 200, R&D Systems) and IRDye 800 conjugated affinity purified anti-mouse IgG (1 : 3000, Rockland Inc., Gilbertsville, PA, USA).

Techniques: Incubation, In Vitro, Purification, Recombinant, SDS Page, Membrane, Western Blot, Staining