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: The Journal of biological chemistry

Article Title: Rapid redistribution of CD20 to a low density detergent-insoluble membrane compartment.

doi: 10.1074/jbc.273.1.344

Figure Lengend Snippet: FIG. 1. FACScan profiles of CD20 mAb binding to Raji cells. Indirect immunofluorescence was performed as described under “Ex- perimental Procedures.” Solid profile represents the binding of the fluorescein isothiocyanate-labeled secondary antibody. Open profiles show binding of the primary antibodies 1F5, 2H7, and B1 as indicated.

Article Snippet: Bands were visualized using Kodak X-OMAT film (Eastman Kodak Co.) Immunofluorescence—Cells (2 3 105) were suspended and incubated in 100 ml of RPMI 1640 medium, 10% fetal bovine serum for 15 min at 37 °C with 2H7, 1F5, B1, or isotype-matched control mAb, washed once, and resuspended for 15 min with 100 ml of 1/100 dilution of goat anti-mouse IgG conjugated to fluorescein isothiocyanate (Southern Biotechnology Associates, Inc.).

Techniques: Binding Assay, Immunofluorescence, Labeling

Journal: The Journal of biological chemistry

Article Title: Rapid redistribution of CD20 to a low density detergent-insoluble membrane compartment.

doi: 10.1074/jbc.273.1.344

Figure Lengend Snippet: FIG. 5. 2H7-induced CD20 redistribution to the Triton-insolu- ble fraction is rapid and does not involve internalization. A, CD20 immunoblot. Cells were treated with 2H7 mAb for the times indicated and lysed immediately with 2 3 lysis buffer. Each lane con- tains Triton-insoluble material from 2 3 105 cells. B, indirect immuno- fluorescence. Solid profile represents the binding of the fluorescein isothiocyanate-labeled secondary antibody. Open profiles, cells were incubated for 15 min at 37 °C with 2H7 and then washed. Fluorescein isothiocyanate-conjugated secondary antibody was either added im- mediately (solid line) or after a further 45-min incubation at 37 °C (dashed line).

Article Snippet: Bands were visualized using Kodak X-OMAT film (Eastman Kodak Co.) Immunofluorescence—Cells (2 3 105) were suspended and incubated in 100 ml of RPMI 1640 medium, 10% fetal bovine serum for 15 min at 37 °C with 2H7, 1F5, B1, or isotype-matched control mAb, washed once, and resuspended for 15 min with 100 ml of 1/100 dilution of goat anti-mouse IgG conjugated to fluorescein isothiocyanate (Southern Biotechnology Associates, Inc.).

Techniques: Western Blot, Lysis, Fluorescence, Binding Assay, Labeling, Incubation

Journal: The Journal of biological chemistry

Article Title: A minimally lipidated form of cell-derived apolipoprotein E exhibits isoform-specific stimulation of neurite outgrowth in the absence of exogenous lipids or lipoproteins.

doi: 10.1074/jbc.273.7.4206

Figure Lengend Snippet: FIG. 2. Western blotting of apoE secreted by stably transfected cell lines. Conditioned media (100 mg of protein) were separated by SDS-10% polyacrylamide gel electrophoresis and were electroblotted to a nitrocellulose membrane. The membrane was probed with an affinity purified polyclonal goat anti-human apoE antibody. After incubation with a horseradish peroxidase-conjugated anti-goat IgG, bands were visualized by enhanced chemiluminescence. The slight difference in mobility of the apoE3 and apoE4 bands is due to curvature in the running front and was not seen in other experiments.

Article Snippet: The cells were washed three times with PBS containing 0.5% Triton X-100 and incubated with a rhodamine-conjugated donkey anti-goat IgG antibody (Jackson Immuno Research) at 1:2000 in in PBS containing 1% FBS and 0.5% Triton X-100 for 1 h at 37 °C.

Techniques: Western Blot, Stable Transfection, Transfection, Polyacrylamide Gel Electrophoresis, Membrane, Affinity Purification, Incubation

Journal: The Journal of biological chemistry

Article Title: A minimally lipidated form of cell-derived apolipoprotein E exhibits isoform-specific stimulation of neurite outgrowth in the absence of exogenous lipids or lipoproteins.

doi: 10.1074/jbc.273.7.4206

Figure Lengend Snippet: FIG. 3. Immunocytochemical detection of apoE in stably transfected cell lines. Neuro-2a parental cells (A and B), Neuro-2a apoE3- secreting cells (C), and Neuro-2a apoE4-secreting cells (D) were grown on glass coverslips. ApoE localization was detected with an affinity purified polyclonal goat anti-human apoE antibody and a rhodamine-conjugated secondary antibody (A, C, and D). The white arrows highlight the concentrated localization of apoE in the growth cone domains in the apoE isoform-expressing Neuro-2a cells. B is the transmitted light image of the parental Neuro-2a cells found in A (black arrows highlight 2 Neuro-2a cells). All images were collected by confocal microscopy with a 40 3 oil immersion lens.

Article Snippet: The cells were washed three times with PBS containing 0.5% Triton X-100 and incubated with a rhodamine-conjugated donkey anti-goat IgG antibody (Jackson Immuno Research) at 1:2000 in in PBS containing 1% FBS and 0.5% Triton X-100 for 1 h at 37 °C.

Techniques: Stable Transfection, Transfection, Affinity Purification, Expressing, Confocal Microscopy