Journal: Molecular Neurodegeneration

Article Title: Evidence against roles for phorbol binding protein Munc13-1, ADAM adaptor Eve-1, or vesicle trafficking phosphoproteins Munc18 or NSF as phospho-state-sensitive modulators of phorbol/PKC-activated Alzheimer APP ectodomain shedding

doi: 10.1186/1750-1326-2-23

Figure Lengend Snippet: Localization of APP following PMA treatment . HEK293 cells were co-transfected with the following cDNAs: (a, b) APP SWE -pPrk5 and Munc13-1 WT -pEGFP-N1 and (c, d) APP SWE -pPrk5 and Munc13-1 H567K -pEGFP-N1. Treatment with 100 nM PMA was carried out for 2 hours. GFP immunofluorescence allowed visualization of (a) Munc13-1 WT and (c) Munc13-1 H567K mutant molecules (green). (b, d) APP was immunolabeled with rabbit polyclonal anti-APP-specific antibody 369 followed by rhodamine red conjugated secondary antibody (red).

Article Snippet: The pEGFP-N1 mammalian expression plasmid was purchased from Clontech.

Techniques: Transfection, Immunofluorescence, Mutagenesis, Immunolabeling

Journal: PLoS ONE

Article Title: SPSB2 inhibits hepatitis C virus replication by targeting NS5A for ubiquitination and degradation

doi: 10.1371/journal.pone.0219989

Figure Lengend Snippet: (A) Myc-SPSB2 was co-transfected with the plasmids coding HCV proteins (core, E1, E2, NS2, NS3/4A, NS4B, NS5A, or NS5B) or pEGFP-C1 (empty vector) for 36 h in HEK293T cells; cell lysates were immunoprecipitated with anti-GFP antibody, and the immunoprecipitates were analyzed by western blot with anti-GFP or anti-Myc antibody. (B) Schematic diagram of the interaction between SPSB2 and HCV proteins. (C) Myc-SPSB2 was co-transfected with HA-NS5A or an empty vector in HEK293T cells for 36 h; cell lysates were immunoprecipitated with anti-HA antibody (a) or anti-Myc antibody (b), and the immunoprecipitates were analyzed by western blot with anti-HA or anti-Myc antibody. (D) Myc-SPSB2 was co-transfected with Flag-E1 or an empty vector in HEK293T cells for 36 h; cell lysates were immunoprecipitated with anti-Flag antibody (a) or anti-Myc antibody (b), and the immunoprecipitates were analyzed by western blot with anti-Flag or anti-Myc antibody. (E) Myc-SPSB2 was co-transfected with HA-NS5A or an empty vector in Huh7 cells for 48 h; cell lysates were immunoprecipitated with anti-HA antibody (a) or anti-Myc antibody (b), and the immunoprecipitates were analyzed by western blot with anti-HA or anti-Myc antibody. (F) Myc-SPSB2 was co-transfected with Flag-E1 or an empty vector in Huh7 cells for 48 h; cell lysates were immunoprecipitated with anti-Flag antibody (a) or anti-Myc antibody (b), and the immunoprecipitates were analyzed by western blot with anti-Flag or anti-Myc antibody. (G) Huh7 cells were transfected with Myc-SPSB2 after infected with HCV JFH-1 (a); Huh7 cells were transfected with Myc-SPSB2 and GFP-E1 (b), and then stained with anti-NS5A antibody 9E10 and FITC-conjugated goat anti-mouse secondary antibody to detect NS5A, as well as anti-Myc antibody and TRITC-conjugated goat anti-rabbit secondary antibody to detect Myc-SPSB2. Cells were stained with DAPI to visualize the nuclei. (H) HCV JFH1-infected Huh7.5.1 cells were harvested after being cultured for 96 h,and lysates were immunoprecipitated with a mouse anti-SPSB2 MAb or control mouse IgG. The immunoprecipitates were subjected to western blot with anti-NS5A (JFH-1) antibody and anti-SPSB2 antibody.

Article Snippet: Viral genes coding HCV proteins were cloned into mammalian expression vectors pEGFP-C1 (Clontech), pRK-7 (Addgene) with a 5′-Flag tag, or pRK-5 (Addgene) with a 5′-HA tag using the template amplified from cDNA of Huh7.5.1 cells infected with HCV JFH-1. pHA-Ub and pMYC-Ub plasmids were kindly provided by Jianguo Wu (College of Life Science, Wuhan University, China).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Infection, Staining, Cell Culture