Journal: The Journal of biological chemistry

Article Title: The plastid-encoded ccsA gene is required for heme attachment to chloroplast c-type cytochromes.

doi: 10.1074/jbc.271.9.4632

Figure Lengend Snippet: FIG. 5. Phenotype of a strain (DccsA) containing a disrupted ccsA gene. Soluble proteins were prepared from copper-supplemented (1Cu) or copper-deficient (2Cu) cultures of the indicated strains and analyzed for the accumulation of holocytochrome c6 (heme stain and anti-cytochrome c6) and plastocyanin (anti-pc). Nitrocellulose membranes were used for the leftmost three panels. Bound antibody was detected by use of a horseradish peroxidase-conjugated secondary antibody. The pellet fractions from the preparation were analyzed for cytochrome f accumulation (anti-cytochrome f). PVDF membranes were used for the transfer, and an alkaline phosphatase-conjugated secondary antibody was used for detection.

Article Snippet: Bound antibodies were detected with alkaline phosphatase- or horseradish peroxidase-conjugated secondary antibody according to instructions provided by the manufacturer (Bio-Rad).

Techniques: Staining

Journal: The Journal of biological chemistry

Article Title: The plastid-encoded ccsA gene is required for heme attachment to chloroplast c-type cytochromes.

doi: 10.1074/jbc.271.9.4632

Figure Lengend Snippet: FIG. 6. Complementation of strain B6 with the cloned ycf5 gene. A, sum- mary of the complementation experi- ments. Plasmids pEBP and pEBH were constructed in vector pTZ19R, while pNH was constructed in the vector pET22b(1). The indicated frequencies are the aver- ages from three different experiments. B, extracts of soluble protein were prepared from copper-deficient cultures of strain CC425 (wt), strain B6, or cells of B6 res- cued by plasmid pEBP (B6-P) or pEBH (B6-H). Equivalent amounts (correspond- ing to 1 A595 unit in the Pierce Coomassie dye binding assay) were analyzed, after separation of proteins in an SDS-contain- ing polyacrylamide gel and transfer to a nitrocellulose membrane, for accumula- tion of holocytochrome c6 by heme stain- ing (bottom panel, 45-min exposure to NEN Reflection film) or decoration with anti-cytochrome c6 (top panel, horse- radish peroxidase conjugated second antibody).

Article Snippet: Bound antibodies were detected with alkaline phosphatase- or horseradish peroxidase-conjugated secondary antibody according to instructions provided by the manufacturer (Bio-Rad).

Techniques: Clone Assay, Construct, Plasmid Preparation, Binding Assay, Membrane, Staining

Journal: Virology

Article Title: Tetherin restricts HSV-2 release and is counteracted by multiple viral glycoproteins.

doi: 10.1016/j.virol.2014.11.005

Figure Lengend Snippet: Fig. 4. Tetherin is highly likely to be incorporated into HSV-2 virions. (A) HSV-2 virion preparations were fractioned via a sucrose gradient cushion (10–50%) to separate cellular membrane fragments and defective viral particles from intact infectious virions. The representative viral and cellular proteins were assessed by western blot. One representative experiment out of three is shown. (B) Immunofluorescence analysis of HSV-2 virions. Virions harvested from HSV-2 infected HeLa cells were pelleted and applied to poly-L-lysine-coated coverslips prior to immunofluorescence analysis using anti-ICP5 and anti-tetherin antibodies. The FITC-positive dots were positive for tetherin and the Cy3-positive dots were positive for HSV-2 ICP5. Representative fields observed in two experiments are shown. Scale bars represent 500 nm.

Article Snippet: Cells were incubated for 1 h at 37 1C with a mouse monoclonal antibody against FLAG (F1804; Sigma) at a dilution of 1:200, gB (mouse monoclonal antibody; ab6506; Abcam) at a dilution of 1:200, gD (mouse monoclonal antibody; sc-58154; Santa Cruz) at a dilution of 1:50 or HSV-2 (sheep polyclonal antibody; PAB13979; Abnova) at a dilution of 1:200, followed by an incubation for 1 h at 37 1C with a FITC-conjugated goat anti-mouse secondary antibody (Boster, China) at a dilution of 1:100, a Cy3-conjugated goat antirabbit secondary antibody (Boster, China) at a dilution of 1:100 or a FITC-conjugated rabbit anti-goat secondary antibody (Boster, China) at a dilution of 1:100 in PBS-2% (w/v) BSA.

Techniques: Membrane, Western Blot, Infection