Journal: The Journal of investigative dermatology

Article Title: Impaired trafficking of the desmoplakins in cultured Darier's disease keratinocytes.

doi: 10.1046/j.1523-1747.2003.12557.x

Figure Lengend Snippet: Figure 6. Dp associates with SERCA2 during di¡erentiation. (A) NHK cells grown in low-calcium medium were pulse-labeled with [35S]methionine and cysteine and then chased in a normal-calcium med- ium for 4 h. Cells were lyzed and immunoprecipitated with the indicated antibody. (B) NHK cells were incubated in normal-calcium medium for 4 h to induce cell di¡erentiation. Cells were lyzed and immunoprecipitated with anti-SERCA2 antibody. SERCA2 immunoprecipitates were analyzed by SDS-PAGE and western blot using an anti-Dp antibody (lane 2). Lane 1, Western blot of total cell extract.

Article Snippet: Mouse monoclonal anti-PG antibody was purchased from Cymbus Bioscience (Southampton, UK), goat polyclonal anti-SERCA2 antibody was from Santa Cruz Biotechnology (Insight Biotechnology, Middlesex, UK), and rabbit polyclonal anti-calnexin antibody was obtained from Calbiochem (CN Biosciences, Nottingham, UK).

Techniques: Labeling, Immunoprecipitation, Incubation, SDS Page, Western Blot

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Amyloid Beta A4 Precursor Protein-binding Family B Member 1 (FE65) Interactomics Revealed Synaptic Vesicle Glycoprotein 2A (SV2A) and Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase 2 (SERCA2) as New Binding Proteins in the Human Brain *

doi: 10.1074/mcp.M113.029280

Figure Lengend Snippet: Novel FE65 interacting protein candidates (significant candidates) (corresponding table in color can be found in supplemental Table S1 )

Article Snippet: The following primary antibodies were used: FE65 (05–759, Millipore), GFP (sc 9996, Santa Cruz Biotechnology Darmstadt, Germany), SV2A (HPA007863, Sigma), and SERCA2 (NB-100–237, Novus Biologicals).

Techniques: Control, Protein Binding, Clinical Proteomics, Membrane, Coagulation

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Amyloid Beta A4 Precursor Protein-binding Family B Member 1 (FE65) Interactomics Revealed Synaptic Vesicle Glycoprotein 2A (SV2A) and Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase 2 (SERCA2) as New Binding Proteins in the Human Brain *

doi: 10.1074/mcp.M113.029280

Figure Lengend Snippet: SV2A, VGLUT1, RYR3, and SERCA2 are novel FE65 interacting proteins. Candidates chosen for further validation from the interaction study were examined in three FE65 (F1–F3) and three control (C1–C3) pulldown assays. A, immunoblotting using the anti-SV2A antibody revealed strong signals corresponding to the full-length (FL) SV2A protein in all F samples that were absent in controls. In addition, a putative SV2A cleavage product (CP) signal was evident in both pulldown eluates and in the brain lysate (BL) used as starting material for the pulldown experiments. B, similarly, VGLUT1 immunoblotting revealed a specific signal in the FE65 precipitates, but only a weak signal in controls (a second brain lysate preparation (BL2) was used in addition to BL). C, a putative RYR3 cleavage product was specifically identified in the F samples, but not in controls. D, finally, immunoblotting for SERCA2 validated our initial findings. A longer exposure (box marked by arrow) as well as an independent experiment (box marked by asterisk) revealed weak signals in the F samples that were not present in the control lanes.

Article Snippet: The following primary antibodies were used: FE65 (05–759, Millipore), GFP (sc 9996, Santa Cruz Biotechnology Darmstadt, Germany), SV2A (HPA007863, Sigma), and SERCA2 (NB-100–237, Novus Biologicals).

Techniques: Biomarker Discovery, Control, Western Blot

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Amyloid Beta A4 Precursor Protein-binding Family B Member 1 (FE65) Interactomics Revealed Synaptic Vesicle Glycoprotein 2A (SV2A) and Sarcoplasmic/Endoplasmic Reticulum Calcium ATPase 2 (SERCA2) as New Binding Proteins in the Human Brain *

doi: 10.1074/mcp.M113.029280

Figure Lengend Snippet: FE65 and SERCA2 co-immunoprecipitate and FE65 knockdown sensitizes cells to the endoplasmic reticulum stressor thapsigargin. A, co-immunoprecipitation assays for FE65 (red) revealed the precipitation of SERCA2 (green) using antibodies against EGFP (IP GFP) or FE65 (IP FE65). FE65 was identified on the SV2A immunoprecipitates (IP SERCA2). Lanes labeled with FE65 and SERCA2 correspond to lysates of cells expressing the individual protein. Lane SN corresponds to the supernatant after immunoprecipitation of a control (without primary antibody) demonstrating the presence of both proteins in the supernatant. B–D, the knockdown of FE65 in HEK293 cells resulted in less cell viability following 8 μm thapsigargin (Tha) stimulation after 24 h (B) and following 3 μm and 8 μm Tha stimulation after 48 h (C). The proteasomal inhibitors epoxomicin (Epoxo) and MG132 revealed no differences in cell viability in FE65 knockdown cells versus controls (D).

Article Snippet: The following primary antibodies were used: FE65 (05–759, Millipore), GFP (sc 9996, Santa Cruz Biotechnology Darmstadt, Germany), SV2A (HPA007863, Sigma), and SERCA2 (NB-100–237, Novus Biologicals).

Techniques: Knockdown, Immunoprecipitation, Labeling, Expressing, Control