Journal: The FASEB Journal

Article Title: Deficient transient receptor potential vanilloid type 4 function contributes to compromised [Ca 2+ ] homeostasis in human autosomal‐dominant polycystic kidney disease cells

doi: 10.1096/fj.201701535rr

Figure Lengend Snippet: Figure 9. TRPV4 deglycosylation is associated with reduced channel activity. A) Summary graph of whole-cell TRPV4- dependent current-voltage (I-V) relations upon channel over- expression in CHO cells in control and after pretreatment with tunicamycin (5 mg/ml) for 24 h to block glycosylation. Currents were induced by the application of hypotonic (220 mOsm) medium from isotonic control values (300 mOsm). B) Representative Western blot of whole-cell lysates in CHO cells that overexpress TRPV4 in control and after tunicamycin treatment. Each line represents individual transfection. Lysates were probed with anti-TRPV4 and anti–b-actin Abs. g, glycosy- lated form of TRPV4.

Article Snippet: TRPV4 expression in Chinese hamster ovary cells Chinese hamster ovary (CHO) cells were obtained from American Type Culture Collection (Manassas, VA, USA).

Techniques: Activity Assay, Over Expression, Control, Blocking Assay, Glycoproteomics, Western Blot, Transfection

Journal: MicrobiologyOpen

Article Title: Identification of regions within the Legionella pneumophila VipA effector protein involved in actin binding and polymerization and in interference with eukaryotic organelle trafficking

doi: 10.1002/mbo3.316

Figure Lengend Snippet: Quantification of co‐localization between VipA‐ EGFP fusion proteins and F‐actin in mammalian CHO cells untreated (−) or treated with cytochalasin D (+cyto). Quantification of co‐localization between VipA and actin was performed by calculating the Manders coefficient using ImageJ and the Plugin JAC oP (see ). The Manders coefficient corresponds to the fraction of VipA‐ EGFP signal (green) overlapping with actin signal (red) divided by the total green signal in a cell. Symbols represent individual cells expressing VipA‐ EGFP mutant proteins ( n ≥ 14 from three independent experiments) and bars indicate mean and standard error of the mean. Statistical analysis was performed as described in and the obtained P value indicating statistically significant differences is indicated (***, P < 0.001). All other differences between data sets are not statistically significant ( P > 0.05).

Article Snippet: For this, we transfected CHO cells with plasmids expressing EGFP (as control), VipA WT ‐EGFP, VipA ΔNH2 ‐EGFP , VipA ΔCC ‐EGFP, VipA ΔCOOH ‐EGFP , or VipA COOH ‐EGFP, and immunoprecipitated the fusion proteins from cell lysates using GFP‐Trap beads (Chromotek).

Techniques: Expressing, Mutagenesis

Journal: MicrobiologyOpen

Article Title: Identification of regions within the Legionella pneumophila VipA effector protein involved in actin binding and polymerization and in interference with eukaryotic organelle trafficking

doi: 10.1002/mbo3.316

Figure Lengend Snippet: Co‐localization between VipA‐ EGFP fusion proteins and EEA 1 in mammalian CHO cells. (A) CHO cells were transfected for 24 h with pEGFP ‐N1 (Clontech) derivatives encoding the indicated proteins. After transfection, cells were fixed and immunolabeled with an anti‐ EEA ‐1 antibody and an appropriate fluorophore‐conjugated secondary antibody. Images shown were collected in a Zeiss LSM 710 confocal microscope. Scale bars, 5 μ m. (B) Quantification of co‐localization between VipA and EEA 1 was performed by calculating the Manders coefficient using ImageJ and the Plugin JAC oP (see ). The Manders coefficient corresponds to the fraction of VipA‐ EGFP signal (green) overlapping with EEA 1 signal (red) divided by the total green signal in a cell. Symbols represent individual cells expressing VipA‐ EGFP mutant proteins ( n ≥ 20, from three independent experiments) and bars indicate mean and standard error of the mean. Statistical analysis was performed as described in Experimental Procedures and the obtained P values indicating statistically significant differences ( P < 0.05) are indicated (**, P < 0.005; ***, P < 0.001).

Article Snippet: For this, we transfected CHO cells with plasmids expressing EGFP (as control), VipA WT ‐EGFP, VipA ΔNH2 ‐EGFP , VipA ΔCC ‐EGFP, VipA ΔCOOH ‐EGFP , or VipA COOH ‐EGFP, and immunoprecipitated the fusion proteins from cell lysates using GFP‐Trap beads (Chromotek).

Techniques: Transfection, Immunolabeling, Microscopy, Expressing, Mutagenesis

Journal: MicrobiologyOpen

Article Title: Identification of regions within the Legionella pneumophila VipA effector protein involved in actin binding and polymerization and in interference with eukaryotic organelle trafficking

doi: 10.1002/mbo3.316

Figure Lengend Snippet: The COOH ‐terminal of VipA is an actin‐binding region. Immunoprecipitation assays of VipA‐ EGFP fusions were carried out with cell extracts from transfected CHO cells using GFP ‐Trap beads (ChromoTek). Immunoblots of the samples corresponding to input fraction (1.5% loaded on gel), nonbound (1.5%), last wash (3%) or bound fractions (60%) were performed with anti‐actin and anti‐ GFP antibodies.

Article Snippet: For this, we transfected CHO cells with plasmids expressing EGFP (as control), VipA WT ‐EGFP, VipA ΔNH2 ‐EGFP , VipA ΔCC ‐EGFP, VipA ΔCOOH ‐EGFP , or VipA COOH ‐EGFP, and immunoprecipitated the fusion proteins from cell lysates using GFP‐Trap beads (Chromotek).

Techniques: Binding Assay, Immunoprecipitation, Transfection, Western Blot