ci1033  (Pfizer Inc)

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Cylindromatosis Tumor Suppressor Protein (CYLD) Deubiquitinase is Necessary for Proper Ubiquitination and Degradation of the Epidermal Growth Factor Receptor *

doi: 10.1074/mcp.M116.066423

Figure Lengend Snippet: CYLD engagement in EGFR signaling. A, CYLD enrichment in the tyrosine-phosphorylated complexes upon EGF stimulation in HeLa cells observed by immunoprecipitation (IP) with antiphosphotyrosine (pTyr) antibodies followed by immunoblotting with indicated antibodies. WCL-whole cell lysate. B, Same as in A, in the presence of the EGFR kinase inhibitors Iressa and CI1033. C, Efficiency of CYLD silencing in HeLa cells. The mRNA (left) and protein (right) levels of CYLD in cells expressing scrambled shRNA (shControl) or shRNA specific to CYLD (shCYLD) as estimated by qPCR and Western blotting, respectively. D, Effect of CYLD silencing on the overall ubiquitination profile of the EGF-activated proteins. Lysates from wild type (WT), shControl and shCYLD HeLa cells were subjected to IP with anti-pTyr antibodies, followed by immunoblotting (IB) with indicated antibodies. See also supplemental Fig. S1.

Article Snippet: EGFR inhibitors (Iressa and CI1033) and Akt inhibitor MK2206 were purchased from Selleckchem (Houston, TX) whereas MEK inhibitor U0126 was from Promega (Madison, WI).

Techniques: Immunoprecipitation, Western Blot, Expressing, shRNA, Ubiquitin Proteomics

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Cylindromatosis Tumor Suppressor Protein (CYLD) Deubiquitinase is Necessary for Proper Ubiquitination and Degradation of the Epidermal Growth Factor Receptor *

doi: 10.1074/mcp.M116.066423

Figure Lengend Snippet: CYLD facilitates ligand-dependent recruitment of Cbl-b to EGFR and subsequent receptor ubiquitination. A, Immunoprecipitation (IP) of EGFR from shControl and shCYLD HeLa cells unstimulated or stimulated with EGF for 6 min, followed by Western blotting with indicated antibodies. WCL-whole cell lysates B, Anti-phosphotyrosine (pTyr) IP from shControl and shCYLD HeLa cells, followed by Western blotting with antibodies against Cbl-b and c-Cbl. C, IP of Cbl-b from shControl and shCYLD HeLa cells, followed by Western blotting with indicated antibodies. Equivalent experiment using anti-GFP antibodies was carried out in parallel as control. D, IP of EGFR from shControl and shCYLD HeLa cells, followed by Western blotting with anti-CYLD antibodies. E, IP of EGFR and Cbl-b from shControl and shCYLD SKOV3 cells (left) or shControl and shCYLD Hep2 cells (right) followed by Western blotting with indicated antibodies.

Article Snippet: EGFR inhibitors (Iressa and CI1033) and Akt inhibitor MK2206 were purchased from Selleckchem (Houston, TX) whereas MEK inhibitor U0126 was from Promega (Madison, WI).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Control

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Cylindromatosis Tumor Suppressor Protein (CYLD) Deubiquitinase is Necessary for Proper Ubiquitination and Degradation of the Epidermal Growth Factor Receptor *

doi: 10.1074/mcp.M116.066423

Figure Lengend Snippet: Impaired EGFR trafficking and degradation in CYLD-silenced cells. A and B, Immunofluorescence images of EGFR (red) in shControl and shCYLD HeLa cells expressing GFP-tagged Rab7 (green) and stimulated with EGF for indicated times. DAPI staining was used to visualize cellular nuclei (blue). Three independent images from the 2 h time point are shown in panel B, where the GFP-Rab7 (green) channel is not shown for better comparison of the EGFR (red) signals. C, Immunoblotting of EGFR on total lysates from shControl and shCYLD cells pre-treated with cycloheximide and stimulated with EGF for indicated times. Three different exposures of the image are shown. D, The mRNA levels of EGFR in shControl and shCYLD cells assessed by qPCR.

Article Snippet: EGFR inhibitors (Iressa and CI1033) and Akt inhibitor MK2206 were purchased from Selleckchem (Houston, TX) whereas MEK inhibitor U0126 was from Promega (Madison, WI).

Techniques: Immunofluorescence, Expressing, Staining, Comparison, Western Blot

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Cylindromatosis Tumor Suppressor Protein (CYLD) Deubiquitinase is Necessary for Proper Ubiquitination and Degradation of the Epidermal Growth Factor Receptor *

doi: 10.1074/mcp.M116.066423

Figure Lengend Snippet: CYLD phosphoTyr15-deficient mutant mimics the effects of CYLD silencing. A, Immunoblotting of whole cell lysates from shControl, shCYLD and shCYLD cells transfected with either Flag-tagged wild type CYLD (CYLD WT) or Flag-tagged CYLD with tyrosine 15-mutated to phenylalanine (CYLD Y15F). B, Immunoprecipitation of Flag-CYLD WT and Flag-CYLD Y15F mutant using anti-Flag antibodies under stringent conditions, followed by immunoblotting with anti-pTyr antibodies. C, Lysates from cells with reconstituted expression of CYLD WT or CYLD Y15F were subjected to immunoprecipitation of EGFR. Precipitated complexes as well as whole cell lysates (WCL) were subjected to immunoblotting with the indicated antibodies. D, Quantitative PRM analysis of Cbl-b immunoprecipitated complexes from CYLD-silenced cells (shCYLD) and cells reconstituted with either CYLD WT or CYLD Y15F mutant. E, Whole cell lysates from shCYLD cells and cells reconstituted with either CYLD WT or CYLD Y15F mutant were subjected to immunoblotting to investigate the degradation of EGFR upon EGF stimulation for the indicated times. F, Quantitative PRM measurements of EGFR levels from the same cells and conditions as in panel E.

Article Snippet: EGFR inhibitors (Iressa and CI1033) and Akt inhibitor MK2206 were purchased from Selleckchem (Houston, TX) whereas MEK inhibitor U0126 was from Promega (Madison, WI).

Techniques: Mutagenesis, Western Blot, Transfection, Immunoprecipitation, Expressing