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1.
FIGURE 5.

FIGURE 5. From: Rab35 and Its GAP EPI64C in T Cells Regulate Receptor Recycling and Immunological Synapse Formation.

Rab35 localization and colocalization with TCR. Confocal images of Jurkat cells transfected with Rab35 alone or with other constructs. A, localization of GFP-Rab35 in transfected Jurkat cells compared with two other Rabs. Arrow highlights Rab35 localization in the pericentriolar region. White arrowheads highlight small vesicles enriched in Rab35, many of which are confirmed to be discontinuous with plasma membrane on serial sections. Red arrowhead highlights peripheral processes. N indicates nucleus. B, colocalization of TCR-ζ mGFP and mRFP-Rab35. Arrows highlight Rab35 and TCR-ζ in the pericentriolar region; white arrowheads highlight small vesicles enriched in Rab35 and TCR-ζ. C, GFP-VAMP3 is localized on many of the Rab35-positive granules (arrowheads) in co-transfected Jurkat cells. N indicates nucleus. Scale bars, 2 μm.

Genaro Patino-Lopez, et al. J Biol Chem. 2008 June 27;283(26):18323-18330.
2.
FIGURE 2.

FIGURE 2. From: Rab35 and Its GAP EPI64C in T Cells Regulate Receptor Recycling and Immunological Synapse Formation.

EPI64C and Rab35 localization and expression. A, Jurkat cells were co-transfected with mRFP-EPI64C and 16 different GFP-tagged Rab proteins. Rab35 was unique in the extent of enrichment on the EPI64C-induced vacuoles compared, for example, to Rab5a and Rab27a. Scale bar, 5 μm. B, Western blot analysis of EPI64C and Rab35 on lysates from a panel of hematopoietic cells and human embryonic kidney (HEK) 293 cells. PBT (peripheral blood T cells) and monocytes are from human blood; Raji is a human Burkitt lymphoma B-cell line.

Genaro Patino-Lopez, et al. J Biol Chem. 2008 June 27;283(26):18323-18330.
3.
FIGURE 4.

FIGURE 4. From: Rab35 and Its GAP EPI64C in T Cells Regulate Receptor Recycling and Immunological Synapse Formation.

EPI64C and Rab35 control receptor recycling. A, EPI64-C and Rab35 DN control transferrin recycling. Jurkat cells transfected with mRFP-tagged EPI64-C, Rab35 DN, or mFRP were loaded with fluorescently labeled transferrin, and Tf recycling to the cell surface was quantified. Cells were gated on the transfected (T) or non-transfected (NT) cells in each population. EPI64C and Rab35DN caused slowdown of recycling compared with RFP and the NT cells from each treatment. Data shown are mean ± S.D. B and C, Jurkat cells were transfected with mRFP-Rab35DN (red) and after 16 h processed for immunofluorescence with anti-TfR (B) or anti-CD3-ε (C) (green). Rab35DN colocalized with both TfR and CD3 on vesicles (arrowheads) and vacuoles (arrows). Scale bars, 2 μm. Note some there is also some diffuse CD3 in ER that is not associated with TfR (38).

Genaro Patino-Lopez, et al. J Biol Chem. 2008 June 27;283(26):18323-18330.
4.
FIGURE 3.

FIGURE 3. From: Rab35 and Its GAP EPI64C in T Cells Regulate Receptor Recycling and Immunological Synapse Formation.

EPI64C is a Rab35 GAP, in vitro and in-cell evidence. EPI64C was analyzed for in vitro GAP activity on bacterially produced Rab35 and in-cell GAP activity. A, kinetic analysis. B, results of densitometric analysis expressed as the amount of the GTP-bound form of the indicated Rab after the reaction (Rab3a, 24 min; Rab27a, 48 min; and Rab35, 48 min) as a percentage of the amount before the reaction. Error bars represent the mean ± S.D. of data from three independent experiments. C, Jurkat cells were transfected with dominant negative constructs Rab35 (S22N) and Rab27a (T23N) and constitutive active constructs Rab35 (Q67L) and Rab27a (Q78L). Only Rab35 dominant negative induced large vacuoles. D, Jurkat cells were transfected with mRFP-EPI64C and Rab35-constitutive active (Q67L) or dominant negative (S22N) constructs. Only the constitutive active prevented EPI64C-induced vacuoles. N indicates nucleus. Scale bars, 2 μm.

Genaro Patino-Lopez, et al. J Biol Chem. 2008 June 27;283(26):18323-18330.
5.
FIGURE 7.

FIGURE 7. From: Rab35 and Its GAP EPI64C in T Cells Regulate Receptor Recycling and Immunological Synapse Formation.

Perturbations of Rab35 or EPI64C impair conjugate formation. A, quantitation of conjugate formation between transfected Jurkat cells and Raji cells in the presence or absence of superantigen SEE assessed by flow cytometry. Labels indicate the construct with which the Jurkat cells were transfected. Values plotted are the mean ± S.E. for results from five independent experiments. **, p < 0.015. B, representative images of Jurkat cells transfected with either dominant negative GFP-Rab35 (S22N) or constitutively active GFP-Rab35 (Q67L) in physical contact with Raji cells (stained with CellTracker Blue) with or without SEE. C, Jurkat cells were transfected with shEPI-A and shEPI-B vectors, and lysates were immunoblotted as indicated. Quantitation is shown of conjugate formation between Jurkat cells (transfected with the indicated construct) and Raji cells in the presence or absence of superantigen SEE assessed by flow cytometry. Values plotted are the mean ± S.E. for results from three independent experiments. **, p < 0.015. D, quantitative scoring of enrichment of TCR at the IS in 25 cells from RFP- and Rab35 DN-transfected Jurkat. Bars represent mean ± S.E. from three independent experiments. **, difference is significant at p = 0.01 by the nonparametric Mann-Whitney test.

Genaro Patino-Lopez, et al. J Biol Chem. 2008 June 27;283(26):18323-18330.
6.
FIGURE 1.

FIGURE 1. From: Rab35 and Its GAP EPI64C in T Cells Regulate Receptor Recycling and Immunological Synapse Formation.

EPI64C induces vesicles containing molecules related to synaptic TCR exocytosis. A, expression of GFP-EPI64C wild type (wt) induces vacuoles in Jurkat cells seen by fluorescence and differential interference contrast, but the GAP-inactivated R141K mutant thereof does not. Images show representative cells 24 h after transfection. N indicates nucleus. B, lucifer yellow (4.0 mg/ml) was added to a suspension of mRFP-EPI64C-transfected cells. After 30 min cells were washed and imaged. C, Jurkat cells were transfected with mRFP-EPI64C and stained with anti-EEA1, anti-LAMP2, anti-calnexin, or anti-TfR, respectively. D, Jurkat cells were cotransfected with mRFP-EPI64C and plasmids encoding GFP-tagged proteins involved in synaptic TCR exocytosis: syntaxin4 (Stx-4), VAMP-3, or TCR-ζ. (Note that Stx-4 localizes not only to plasma membrane but also to endosomal membranes) (31). Scale bars, 2 μm.

Genaro Patino-Lopez, et al. J Biol Chem. 2008 June 27;283(26):18323-18330.
7.
FIGURE 6.

FIGURE 6. From: Rab35 and Its GAP EPI64C in T Cells Regulate Receptor Recycling and Immunological Synapse Formation.

Rab35 localizes to the IS. Confocal images of Rab35-transfected Jurkat cells after incubation with Raji APCs in the absence or presence of superantigen SEE to stimulate conjugate formation. A, representative images of enhanced GFP-Rab35-transfected Jurkat cells in physical contact with Raji cells (stained with Cell-Tracker Blue) with or without superantigen SEE. B, single color, merged, and differential interference contrast images of a representative conjugate between mRFP-Rab35 and TCR-ζ-GFP-transfected Jurkat cells and Raji cells in the presence of SEE. N indicates nucleus. Scale bar, 2 μm. C, transfected T cells were dropped onto SEE superantigen-pulsed Raji B cells (approximate location indicated by dashed circle). Maximum intensity projections from Z-stacks (17 slices, 1 μm apart) were compiled at the indicated time points. The zero time point represents the initiation of imaging, which was prior to the first contact between the T cell and the B cell. Scale bar, 5 μm. (Supplemental movie shows complete image set of maximum intensity projections from this observation).

Genaro Patino-Lopez, et al. J Biol Chem. 2008 June 27;283(26):18323-18330.

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