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: Molecular endocrinology (Baltimore, Md.)

Article Title: N-glycosylation of the prolactin receptor is not required for activation of gene transcription but is crucial for its cell surface targeting.

doi: 10.1210/mend.12.4.0085

Figure Lengend Snippet: Fig. 4. Perinuclear Localization of the Aglycosylated PRLR COS-7 cells transfected with WT (a and d), N80,108D (b and e), or N35,80,108D (c and f) PRLR cDNAs were fixed in 4% paraformaldehyde (a, b, and c: nonpermeabilized conditions) or in methanol, 220 C (d, e, and f: permeabilized conditions). Mouse monoclonal antibody U5 (160 mg/ml) followed by FITC goat anti-IgG (dilution 1:40) were used, as described in Materials and Methods. Note the immunostaining in the juxtanuclear area of the aglycosylated PRLR in permeabilized cells (f) and the absence of expression at the plasma membrane level in nonpermeabilized cells (c). 293 cells transfected with WT (g), N80,108D (h), or N35,80,108D (i) PRLR cDNAs were processed for fixation (4% paraformaldehyde) and immunofluorescence staining, as described for COS-7 cells. Note the absence of immunostaining at the cell surface and low perinuclear staining of the aglycosylated receptor in nonpermeabilized conditions (i). Magnification, 3400.

Article Snippet: The secondary antibodies used in these studies were the Texas-Red-conjugated and fluorescein-isothiocyanate-conjugated (FITC) goat anti-mouse IgG (dilution 1:40) (Vector Laboratories, Burlingame, CA; and Biosys, Compiègne, France), the biotinylated donkey anti-rabbit IgG and the Texas-Red-conjugated streptavidin (1:100; Amersham International, Aylesbury, UK).

Techniques: Transfection, Immunostaining, Expressing, Clinical Proteomics, Membrane, Immunofluorescence, Staining

Journal: Molecular endocrinology (Baltimore, Md.)

Article Title: N-glycosylation of the prolactin receptor is not required for activation of gene transcription but is crucial for its cell surface targeting.

doi: 10.1210/mend.12.4.0085

Figure Lengend Snippet: Fig. 5. Deglycosylated PRLR Colocalizes with the Golgi rab6 in COS-7 Cells a, Labeling of fixed permeabilized COS-7 cells transfected with N35,80,108D receptor using U5 antibody. Panels b and c and panels d and e show a double- labeling experiment of fixed permeabilized COS-7 cells transfected with the N35,80,108D mutant, with anti-PRLR antibody [visualized with a secondary anti-mouse FITC secondary antibody (b and d)], and anti-Golgi antibody [anti-rab6 visualized with bioti- nylated secondary anti-rabbit IgG followed by Texas-Red- conjugated streptavidin (c and e)]. The choice of mouse anti- PRLR and rabbit anti-Golgi IgG as primary antibodies allows accurate double-labeling experiments. Panels b-c and d-e show two different COS-7 cells. f, Micrographs of COS-7 cells stained for anti-protein disulfide isomerase (endoplas- mic reticulum protein) antibody, visualized with Texas-Red- conjugated anti-mouse secondary antibody. Magnification, 3400.

Article Snippet: The secondary antibodies used in these studies were the Texas-Red-conjugated and fluorescein-isothiocyanate-conjugated (FITC) goat anti-mouse IgG (dilution 1:40) (Vector Laboratories, Burlingame, CA; and Biosys, Compiègne, France), the biotinylated donkey anti-rabbit IgG and the Texas-Red-conjugated streptavidin (1:100; Amersham International, Aylesbury, UK).

Techniques: Labeling, Transfection, Mutagenesis, Staining