Journal: Molecular & Cellular Proteomics

Article Title: Quantitative Proteomics Identifies a β-Catenin Network as an Element of the Signaling Response to Frizzled-8 Protein-Related Antiproliferative Factor

doi: 10.1074/mcp.m110.007492

Figure Lengend Snippet: FIG. 1. SILAC-based quantitative proteomics analysis identifies APF-responsive proteins. A, Diagram describing our approach toward minimizing false discoveries caused by differential SILAC labeling and/or arginine-to-proline conversion. APF and mock APF (abbreviated as Mock) were purified from the supernatant of bladder epithelial cells explanted from IC patients and healthy controls, respectively, as described (1). T24 cells cultured in “heavy” (“H”) medium were treated with mock APF peptide and used as an internal standard so that the levels of proteins extracted from APF- or mock APF-treated cells, both cultured in “light” (“L”) medium, can be quantitatively compared. B, SDS-PAGE gel image of two samples of “heavy” and “light” protein mixture. Each gel lane was cut into 7 slices with approximately the same length. C, A representative tandem mass spectrum derived from the analysis of a doubly charged peptide (m/z 930.96) from PKM2. D, Representative SILAC pairs showing that COX-2 (i.e. PTGS2) was up-regulated by APF treatment and unchanged by differential SILAC labeling and/or arginine-to-proline conversion.

Article Snippet: The human bladder cancer T24 cell line was obtained from the American Type Culture Collection.

Techniques: Multiplex sample analysis, Quantitative Proteomics, Labeling, Purification, Cell Culture, SDS Page, Derivative Assay

Journal: Molecular & Cellular Proteomics

Article Title: Quantitative Proteomics Identifies a β-Catenin Network as an Element of the Signaling Response to Frizzled-8 Protein-Related Antiproliferative Factor

doi: 10.1074/mcp.m110.007492

Figure Lengend Snippet: FIG. 3. APF induces the down-regulation of -catenin. A, Western blot analysis shows that APF treatment resulted in the down-regulation of total, nuclear, as well as membrane-associated -catenin. T24 cells were treated with APF peptide or mock APF peptide (abbreviated as Mock) for 3 days. Total cell lysates, nuclear extracts and membrane fractions were prepared for immunoblot- ting. -actin, lamin A/C, or caveolin-1 was used as a loading control for whole-cell lysates, nuclear proteins, or membrane proteins, respectively. B, Immunofluorescence analysis shows that APF treatment led to the downmodulation of nuclear and plasma mem- brane-associated -catenin. T24 cells were treated with APF or mock APF for 6 hours prior to labeling cells with anti--catenin primary antibody and Cy3.5 conjugated secondary antibody. Nuclei were stained with DAPI.

Article Snippet: The human bladder cancer T24 cell line was obtained from the American Type Culture Collection.

Techniques: Western Blot, Membrane, Control, Immunofluorescence, Clinical Proteomics, Labeling, Staining

Journal: Molecular & Cellular Proteomics

Article Title: Quantitative Proteomics Identifies a β-Catenin Network as an Element of the Signaling Response to Frizzled-8 Protein-Related Antiproliferative Factor

doi: 10.1074/mcp.m110.007492

Figure Lengend Snippet: FIG. 4. APF treatment induces -catenin internalization and degradation. A, Internalization of -catenin in response to APF. Immuno- fluorescence staining for -catenin in T24 cells was performed 6 hours after treatment with APF or mock APF. -catenin was labeled with anti--catenin primary antibody and Cy3.5 conjugated secondary antibody. B, A pool of -catenin localizes to proteasomes (upper panels) and lysosomes (lower panels) after APF treatment. T24 cells were fixed 6 hours after APF treatment and stained with specific antibodies against -catenin and proteasome subunit Rpt1, lysosomal probe LysoTracker, or DAPI. C, Inhibition of proteasomal and lysosomal degradation reduces -catenin degradation, resulting in higher levels of -catenin. Pretreatment of T24 cells with 10 M MG132, a specific and potent proteasome inhibitor, or 100 g/ml leupeptin, which inhibits lysosomal cysteine proteases, suppressed -catenin degradation in the presence of APF. Blots are representative of at least three independent experiments.

Article Snippet: The human bladder cancer T24 cell line was obtained from the American Type Culture Collection.

Techniques: Fluorescence, Staining, Labeling, Inhibition

Journal: Molecular & Cellular Proteomics

Article Title: Quantitative Proteomics Identifies a β-Catenin Network as an Element of the Signaling Response to Frizzled-8 Protein-Related Antiproliferative Factor

doi: 10.1074/mcp.m110.007492

Figure Lengend Snippet: FIG. 5. Gene silencing of -catenin inhibits cell proliferation whereas overexpressing nondegradable -catenin rescues growth inhibition in response to APF. A, Gene silencing of -catenin suppresses cell proliferation. -catenin level was down-regulated using gene silencing with siRNA transfection. To confirm the knockdown of -catenin, whole-cell lysates from T24 cells nontransfected (abbreviated as NT Ctrl), transfected with control siRNAs (siCtrl), or transfected with -catenin siRNAs (si-catenin) were blotted with anti--catenin antibody. Results presented here are representative of three independent experiments. Cell proliferation rate was measured using crystal violet staining. B, Overexpression of nondegradable -catenin (ND -catenin) rescues cell proliferation inhibition caused by APF treatment. A nondegradable -catenin construct (or vector only) was expressed in T24 cells. 36 hours after transfection, cells were treated with APF or mock APF peptides for an additional 3 days. Expression levels of -catenin protein were determined using Western blotting. Cell proliferation rate was measured using the crystal violet assay.

Article Snippet: The human bladder cancer T24 cell line was obtained from the American Type Culture Collection.

Techniques: Inhibition, Transfection, Knockdown, Control, Staining, Over Expression, Construct, Plasmid Preparation, Expressing, Western Blot, Crystal Violet Assay