Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Surface expression of the IFN-gamma R2 chain is regulated by intracellular trafficking in human T lymphocytes.

doi: 10.4049/jimmunol.164.1.201

Figure Lengend Snippet: FIGURE 1. Flow cytometry of surface and cytoplasmic expression of IFN-gR2. Surface (A, left panel) and cytoplasmic (A, right panel) expres- sion of resting CD31 PBL, CD31 PBL stimulated for 24 h with PHA, and T lymphoblasts cultured with IL-2 for 5, 10, and 15 days. Histograms represent the IFN-gR2 expression of unpermeabilized or permeabilized CD31-gated cells. The background fluorescence detected with FITC-con- jugated isotype-matched control Ig on fresh CD31 T lymphocytes is indi- cated by black profiles. Only the background fluorescence histograms of CD31 fresh T lymphocytes are shown, because the background fluores- cences of CD31 lymphocytes recovered at different times of PHA activa- tion were similar. Each experiment represents the results from six donors. B, T lymphoblasts were cultured in complete medium containing monensin for 4 h with (lower panels) or without (upper panels) PHA, PMA, and iono- mycin. Cells were then stained intracellularly to determine the presence of IFN-g and IFN-gR2 (right panels). Left panels show the background fluores- cence detected with PE- and FITC-isotype matched control Ig.

Article Snippet: RPMI 1640, FCS, L-glutamine, penicillin, streptomycin, gentamicin, and trypan blue dye were from Life Technologies (Grand Island, NY); PHA, PMA, ionomycin, paraformaldehyde, 2-ME, EDTA, HEPES, PMSF, DTT, pepstatin, aprotinin, leupeptin, benzamidine, glycerol, paraformaldehyde, propidium iodide, Tris, RNase A, Tween 20, monensin, saponin, and FITC were from Sigma (St. Louis, MO); DMSO, PBS, BSA, MgCl2, and sodium azide were from Merck (Milan, Italy); KCl, NaCl, HCl, and ammonium peroxodisulfate were from Fluka (Milan, Italy); SDS, acrylamide, bisacrylamide, and N, N, N9, N9-tetramethylethylenediamine were from Bio-Rad (Rockland, ME); PE-conjugated mouse anti-human CD3 mAb, isotype control FITC-conjugated mouse IgG2a, FITC-conjugated mouse IgG1, and PE-conjugated mouse IgG1 were from Dako (Milan, Italy); rabbit polyclonal anti-IRF-1 Ab was from Santa Cruz Biotechnology (Santa Cruz, CA); HRP-conjugated goat anti-rabbit IgG was from Amersham International (Little Chalfont, U.K.); PE-conjugated mouse IgG1 anti-IFN-g and *Department of Clinical and Biological Sciences, University of Turin, Orbassano, Italy; †Department of Experimental Medicine and Pathology, “La Sapienza” University, Rome, Italy; and ‡Hôpital Necker-Enfants Malades, Institut National de la Santé et de la Recherche Médicale, Unité 429, Paris, France Received for publication May 26, 1999.

Techniques: Flow Cytometry, Expressing, Cell Culture, Control, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Surface expression of the IFN-gamma R2 chain is regulated by intracellular trafficking in human T lymphocytes.

doi: 10.4049/jimmunol.164.1.201

Figure Lengend Snippet: FIGURE 4. Flow cytometry of IFN-gR2 and CTLA-4 internalization in T lymphoblasts. A, T lymphoblasts incubated with C.11-FITC (upper pan- els) or PE-anti-CTLA-4 mAb (lower panels) for 4 h at 4°C (left panels) or at 37°C (right panels) were evaluated by flow cytometric analysis as de- scribed in Materials and Methods. Bold lines represent specific fluores- cence with specific mAb, whereas thin lines represent background fluores- cence of FITC- or PE-conjugated isotype-matched mAb. B, time course of C.11-FITC (left panel) or PE-anti-CTLA-4 mAb (right panel) uptake dur- ing incubation at 37°C. Mean specific fluorescence was calculated by sub- tracting the mean of fluorescence obtained with specific mAb to that ob- tained with isotype-matched mAb.

Article Snippet: RPMI 1640, FCS, L-glutamine, penicillin, streptomycin, gentamicin, and trypan blue dye were from Life Technologies (Grand Island, NY); PHA, PMA, ionomycin, paraformaldehyde, 2-ME, EDTA, HEPES, PMSF, DTT, pepstatin, aprotinin, leupeptin, benzamidine, glycerol, paraformaldehyde, propidium iodide, Tris, RNase A, Tween 20, monensin, saponin, and FITC were from Sigma (St. Louis, MO); DMSO, PBS, BSA, MgCl2, and sodium azide were from Merck (Milan, Italy); KCl, NaCl, HCl, and ammonium peroxodisulfate were from Fluka (Milan, Italy); SDS, acrylamide, bisacrylamide, and N, N, N9, N9-tetramethylethylenediamine were from Bio-Rad (Rockland, ME); PE-conjugated mouse anti-human CD3 mAb, isotype control FITC-conjugated mouse IgG2a, FITC-conjugated mouse IgG1, and PE-conjugated mouse IgG1 were from Dako (Milan, Italy); rabbit polyclonal anti-IRF-1 Ab was from Santa Cruz Biotechnology (Santa Cruz, CA); HRP-conjugated goat anti-rabbit IgG was from Amersham International (Little Chalfont, U.K.); PE-conjugated mouse IgG1 anti-IFN-g and *Department of Clinical and Biological Sciences, University of Turin, Orbassano, Italy; †Department of Experimental Medicine and Pathology, “La Sapienza” University, Rome, Italy; and ‡Hôpital Necker-Enfants Malades, Institut National de la Santé et de la Recherche Médicale, Unité 429, Paris, France Received for publication May 26, 1999.

Techniques: Flow Cytometry, Incubation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Surface expression of the IFN-gamma R2 chain is regulated by intracellular trafficking in human T lymphocytes.

doi: 10.4049/jimmunol.164.1.201

Figure Lengend Snippet: FIGURE 5. Localization of IFN-gR2 on the surface and in the cyto- plasm of T lymphoblasts. Serial optical sections 0.5 mm apart through cells incubated for 4 h with C.11-FITC at 4°C (upper panel) and at 37°C (lower panel). Nuclear DNA was stained with propidium iodide (red fluores- cence); green fluorescence localizes the cytoplasmic vesicles containing C.11-FITC.

Article Snippet: RPMI 1640, FCS, L-glutamine, penicillin, streptomycin, gentamicin, and trypan blue dye were from Life Technologies (Grand Island, NY); PHA, PMA, ionomycin, paraformaldehyde, 2-ME, EDTA, HEPES, PMSF, DTT, pepstatin, aprotinin, leupeptin, benzamidine, glycerol, paraformaldehyde, propidium iodide, Tris, RNase A, Tween 20, monensin, saponin, and FITC were from Sigma (St. Louis, MO); DMSO, PBS, BSA, MgCl2, and sodium azide were from Merck (Milan, Italy); KCl, NaCl, HCl, and ammonium peroxodisulfate were from Fluka (Milan, Italy); SDS, acrylamide, bisacrylamide, and N, N, N9, N9-tetramethylethylenediamine were from Bio-Rad (Rockland, ME); PE-conjugated mouse anti-human CD3 mAb, isotype control FITC-conjugated mouse IgG2a, FITC-conjugated mouse IgG1, and PE-conjugated mouse IgG1 were from Dako (Milan, Italy); rabbit polyclonal anti-IRF-1 Ab was from Santa Cruz Biotechnology (Santa Cruz, CA); HRP-conjugated goat anti-rabbit IgG was from Amersham International (Little Chalfont, U.K.); PE-conjugated mouse IgG1 anti-IFN-g and *Department of Clinical and Biological Sciences, University of Turin, Orbassano, Italy; †Department of Experimental Medicine and Pathology, “La Sapienza” University, Rome, Italy; and ‡Hôpital Necker-Enfants Malades, Institut National de la Santé et de la Recherche Médicale, Unité 429, Paris, France Received for publication May 26, 1999.

Techniques: Incubation, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Surface expression of the IFN-gamma R2 chain is regulated by intracellular trafficking in human T lymphocytes.

doi: 10.4049/jimmunol.164.1.201

Figure Lengend Snippet: FIGURE 6. Co-localization of IFN-gR2 and CTLA-4 in endocytic ves- icles. T lymphoblasts were incubated with PE-anti-CTLA-4 mAb (red flu- orescence) for 3 h at 37°C. Then cells were washed, fixed, and permeabil- ized, and their internal IFN-gR2 stores were stained with C.11-FITC (green fluorescence). Cells were fixed again and examined by confocal micros- copy. Areas of coincidence of red and green fluorescence (giving light blue fluorescence) indicate overlapping distribution of IFN-gR2 and CTLA-4.

Article Snippet: RPMI 1640, FCS, L-glutamine, penicillin, streptomycin, gentamicin, and trypan blue dye were from Life Technologies (Grand Island, NY); PHA, PMA, ionomycin, paraformaldehyde, 2-ME, EDTA, HEPES, PMSF, DTT, pepstatin, aprotinin, leupeptin, benzamidine, glycerol, paraformaldehyde, propidium iodide, Tris, RNase A, Tween 20, monensin, saponin, and FITC were from Sigma (St. Louis, MO); DMSO, PBS, BSA, MgCl2, and sodium azide were from Merck (Milan, Italy); KCl, NaCl, HCl, and ammonium peroxodisulfate were from Fluka (Milan, Italy); SDS, acrylamide, bisacrylamide, and N, N, N9, N9-tetramethylethylenediamine were from Bio-Rad (Rockland, ME); PE-conjugated mouse anti-human CD3 mAb, isotype control FITC-conjugated mouse IgG2a, FITC-conjugated mouse IgG1, and PE-conjugated mouse IgG1 were from Dako (Milan, Italy); rabbit polyclonal anti-IRF-1 Ab was from Santa Cruz Biotechnology (Santa Cruz, CA); HRP-conjugated goat anti-rabbit IgG was from Amersham International (Little Chalfont, U.K.); PE-conjugated mouse IgG1 anti-IFN-g and *Department of Clinical and Biological Sciences, University of Turin, Orbassano, Italy; †Department of Experimental Medicine and Pathology, “La Sapienza” University, Rome, Italy; and ‡Hôpital Necker-Enfants Malades, Institut National de la Santé et de la Recherche Médicale, Unité 429, Paris, France Received for publication May 26, 1999.

Techniques: Incubation, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Surface expression of the IFN-gamma R2 chain is regulated by intracellular trafficking in human T lymphocytes.

doi: 10.4049/jimmunol.164.1.201

Figure Lengend Snippet: FIGURE 7. Kinetics of IFN-gR2 and CTLA-4 internalization in T lym- phoblasts cultured with or without potassium. T lymphoblasts were cul- tured in medium with (f) or without (E) potassium in the presence of C.11-FITC or PE-anti-CTLA-4 mAb at 37°C as described in Materials and Methods. Time-dependent endocytosis of both mAb was measured by flow cytometry. Relative fluorescence was evaluated as described in the legend of Fig. 4.

Article Snippet: RPMI 1640, FCS, L-glutamine, penicillin, streptomycin, gentamicin, and trypan blue dye were from Life Technologies (Grand Island, NY); PHA, PMA, ionomycin, paraformaldehyde, 2-ME, EDTA, HEPES, PMSF, DTT, pepstatin, aprotinin, leupeptin, benzamidine, glycerol, paraformaldehyde, propidium iodide, Tris, RNase A, Tween 20, monensin, saponin, and FITC were from Sigma (St. Louis, MO); DMSO, PBS, BSA, MgCl2, and sodium azide were from Merck (Milan, Italy); KCl, NaCl, HCl, and ammonium peroxodisulfate were from Fluka (Milan, Italy); SDS, acrylamide, bisacrylamide, and N, N, N9, N9-tetramethylethylenediamine were from Bio-Rad (Rockland, ME); PE-conjugated mouse anti-human CD3 mAb, isotype control FITC-conjugated mouse IgG2a, FITC-conjugated mouse IgG1, and PE-conjugated mouse IgG1 were from Dako (Milan, Italy); rabbit polyclonal anti-IRF-1 Ab was from Santa Cruz Biotechnology (Santa Cruz, CA); HRP-conjugated goat anti-rabbit IgG was from Amersham International (Little Chalfont, U.K.); PE-conjugated mouse IgG1 anti-IFN-g and *Department of Clinical and Biological Sciences, University of Turin, Orbassano, Italy; †Department of Experimental Medicine and Pathology, “La Sapienza” University, Rome, Italy; and ‡Hôpital Necker-Enfants Malades, Institut National de la Santé et de la Recherche Médicale, Unité 429, Paris, France Received for publication May 26, 1999.

Techniques: Cell Culture, Cytometry

Journal: The Journal of Physiology

Article Title: Ectopic expression of cone-specific G-protein-coupled receptor kinase GRK7 in zebrafish rods leads to lower photosensitivity and altered responses

doi: 10.1113/jphysiol.2010.204156

Figure Lengend Snippet: A, schematic diagram of the transgene introduced into the GRK7- or GRK1-tg lines. B, retinal sections of adult wild-type and GRK7-tg fish were immunostained with anti-carp GRK7 antibody; scale bar, 20 μm. C and D, expression levels of GRK7 in GRK7-tg eyes, and of GRK1 in GRK7-tg and GRK1-tg eyes, were assessed by immunoblot analysis of serial dilutions of ocular homogenates from age-matched wild-type and transgenic zebrafish. The amount of protein loaded onto each lane is indicated. His-HA-tagged recombinant GRK proteins, His-HA-GRK7–1 and His-HA-GRK1A, (Wada et al. 2006), were used as standards for the calibration of ocular GRK protein levels. The ocular homogenates and His-HA-tagged recombinant GRK proteins were mixed with zebrafish brain homogenate to adjust the total amounts of proteins to the same level so as to eliminate any difference between the samples in masking effects of non-GRK proteins. Note that the zebrafish brain homogenate showed no detectable immunoreactivities to the primary antibodies against GRKs. Typical immunoblot images are shown in C. The estimated amounts of both GRK1 and GRK7 proteins per total ocular protein are shown in D. Averages (also indicated as numbers) from three independent experiments are shown with SEM. Note that the GRK1 levels (left panel in D) contain GRK1B, which is expressed in cones (Wada et al. 2006). The GRK1B levels are estimated to be 0.018 ng per μg of total protein according to Wada et al. (2006). On the other hand, the GRK7 levels in GRK7-tg (right panel in D) contain GRK7 intrinsic to cones, which is estimated to be 0.15 ng per μg of total protein. After subtracting the amounts of GRK1B and GRK7 in the cones, the ratio of GRK7:GRK1 expression in the rods was estimated as 10:1 for each of the three lines of GRK7-tg animal. E, rhodopsin content in ocular membrane extracts estimated by spectrophotometric measurement. Averages from 10 wild-type fish, five GRK7-tg fish and four GRK1-tg fish are shown with SEM. F and G, expression levels of rod transducin α subunit (GNAT1, F) and rod arrestin (Arr1, G) in ocular homogenates. Top, typical immunoblot images from wild-type and transgenic zebrafish. Bottom, protein expression levels in the eyes of transgenic fish relative to those of wild-type. In each lane, ocular homogenate containing 3.2 μg of total protein was loaded. Averages from four to six independent experiments are shown with SEM.

Article Snippet: The membrane was then incubated with one of the following primary antibodies in the blocking solution overnight at 4°C: anti-carp GRK7 antibody, 1:1000 dilution ( Tachibanaki et al. 2005 ); anti-GRK1 antibody (0.2 μg ml −1 ; sc-8004, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); anti-GNAT1 antibody (0.2 μg ml −1 ; sc-389, Santa Cruz Biotechnology); anti-carp Arr1 antiserum, 1:2000; anti-carp GC-R1 antiserum,1:400 ( Takemoto et al. 2009 ); anti-frog S-modulin antiserum, 1:400 ( Arinobu et al. 2010 ); or anti-carp RGS9 antiserum, 1:400 (S. Tachibanaki, S. Yonetsu, S. Fukaya & S. Kawamura, unpublished observations).

Techniques: Expressing, Western Blot, Transgenic Assay, Recombinant, Membrane