human leukemic t cell lines jurkat (e6-1)  (CEM Corporation)

Journal: bioRxiv

Article Title: Polyubiquitin-dependent recruitment of NEMO/IKKγ into T cell receptor signaling microclusters

doi: 10.1101/617126

Figure Lengend Snippet: A) Jurkat T cells stably transduced with mYFP.NEMO were activated on stimulatory glass surfaces coated with 10 μg/ml anti-CD3 (OKT3), 1 μg/ml rhVCAM1 and/or 10 μg/ml fibronectin, as indicated. Dotted blue arrows, marked ‘P’, identify perinuclear pools of NEMO. Objects marked with a red ‘M’ are typical of the large objects referred to as ‘macroclusters’. Similar patterns were observed in every experiment (n≥3 for each condition). Scale bars: 10 μm. B) Primary human T cell blasts were transduced, stimulated, and imaged as in A. Images are representative of four experiments. C) The lag between local contact initiation and NEMO microcluster formation was calculated for cells captured in the process of spreading. Data are presented as the mean ± SD based on the number of cells examined. 468 clusters were observed in thirteen cells stimulated on anti-CD3 (3, n=8 cells) or anti-CD3 and rhVCAM1 (3-V, n=2 cells). No significant differences were observed between any group of cells and the pool of all conditions, or the cells stimulated on anti-CD3. D) Jurkat T cells stably transduced with mYFP.NEMO were stimulated on anti-CD3 and rhVCAM1 and fixed after five minutes. NEMO clusters were identified algorithmically (see Materials and Methods). The resulting masks are pseudo-colored (green) and superimposed on the raw image (red). Line scans show NEMO intensity (red line) along the indicated white line relative to the cluster masks (shaded green). E) Jurkat T cells were transiently transfected or stably transduced with mYFP.NEMO and imaged on the indicated substrates. NEMO clusters identified as in D were binned into classes based on cluster area. Classes are labeled using the diameter of a circle with an area equivalent to the upper bound of the class. Graphs present the cluster count (upper), the fractional distribution of clusters by class (middle), and the fraction of the NEMO intensity in the imaging plane that is captured within each class (lower). Data are presented as the mean ± SEM, based on the number of cells analyzed. Cluster quantitation was performed using 23 cells stimulated on anti-CD3 and rhVCAM1 and 13 cells stimulated on rhVCAM1 alone. Statistical differences among classes were determined using Student’s T-test: p < 0.05, *; p < 0.01, **; p < 0.001, ***. F) Jurkat cells expressing mYFP.NEMO and NFκB-luciferase plus either mCer3 itself or mCer3 chimeras with WT or kinase-deficient (KR) IKKβ were stimulated as indicated; levels of luciferase were measured to indicate the amount of NF-κB transcription. G) Jurkat T cells stably expressing mYFP.NEMO WT and either mCer3.IKKβ WT or mCer3.IKKβ-K44R (kinase-deficient mutant). Kymographs for each were taken from the region in the white box, and represent 5 minutes in time. H) Model showing the location of NEMO S68 in the context of a NEMO dimer; phosphorylation of this site is predicted to cause destabilization of the IKK complex and dissolution of the NEMO/ IKKβ microcluster.

Article Snippet: The Jurkat human leukemic T cell line E6.1 (TIB-152; RRID:CVCL_0367) and the C305 hybridoma (CRL-2424; RRID:CVCL_K130) were obtained from ATCC (Manassas, VA).

Techniques: Stable Transfection, Transduction, Transfection, Labeling, Imaging, Quantitation Assay, Expressing, Luciferase, Mutagenesis, Phospho-proteomics, Dissolution

Journal: bioRxiv

Article Title: Polyubiquitin-dependent recruitment of NEMO/IKKγ into T cell receptor signaling microclusters

doi: 10.1101/617126

Figure Lengend Snippet: A) SLP76-deficient J14 Jurkat cells stably reconstituted with SLP-76.YFP, parental Jurkat T cells and primary human T cell blasts were transiently transfected as indicated and stimulated on anti-CD3, rhVCAM1, and anti-CD28, as in . Images were pseudo-colored as indicated. The regions enclosed in magenta boxes are enlarged at right. Relative fluorescence intensities along the white diagonal lines are shown in the lower panels. Data are representative of three-six experiments for Jurkat cells and two experiments for primary cells. B) Jurkat T cells were transiently transfected with mYFP.NEMO and ZAP-70.mCFP, stimulated on anti-CD3, and imaged over time. A pseudo-colored still image is shown at left. Blue arrows identify a perinuclear pool that is not anchored to the substrate. The regions enclosed in magenta boxes were used to generate the kymographs shown at right. White arrows identify points at which static NEMO clusters begin to move, and asterisks identify the points at which mobile NEMO clusters stop. Still images are representative of six experiments. Scale bars: 10 μm (stills); 2 μm (insets); 5 μm × 60 seconds (kymographs). C) Graphs present the fractional distribution of NEMO clusters by size class (left), the fraction of total ZAP intensity within each NEMO class (center-left), the fraction of the total area masked as a ZAP cluster that lies within each NEMO class (center), the per-pixel enrichment of ZAP intensity within each NEMO class, relative to the entire cell (center-right), and the per-pixel enrichment of ZAP masked area within each NEMO class, relative to the entire cell (right). Statistical differences among corresponding classes were determined using Student’s T-test: p < 0.05, *; p < 0.01, **; p < 0.001, ***. Data are presented as the mean ±SEM, based on the number of cells analyzed. Calculations were performed for 12 cells co-expressing NEMO and ZAP-70.

Article Snippet: The Jurkat human leukemic T cell line E6.1 (TIB-152; RRID:CVCL_0367) and the C305 hybridoma (CRL-2424; RRID:CVCL_K130) were obtained from ATCC (Manassas, VA).

Techniques: Stable Transfection, Transfection, Fluorescence, Expressing

Journal: bioRxiv

Article Title: Polyubiquitin-dependent recruitment of NEMO/IKKγ into T cell receptor signaling microclusters

doi: 10.1101/617126

Figure Lengend Snippet: A) Diagram of NEMO domain structure and truncations used in subsequent experiments. B) NEMO-deficient 8321 cells expressing an NF-κB-driven rat Thy1 reporter were transiently transfected with vectors expressing mYFP alone, or mYFP.NEMO chimeras bearing the indicated deletions. After stimulation with PMA, plus either ionomycin or anti-CD28, these cells were analyzed by flow cytometry. Thy1 MFI is shown for cells expressing comparable levels of mYFP. Statistical differences were determined using Student’s T-test: p < 0.01, **; p < 0.001, ***. Data are presented as the mean ± SD of three technical replicates and are representative of 2-5 experiments. C) Jurkat T cells stably expressing wild-type or truncated mYFP.NEMO chimeras were stimulated as in and fixed after five minutes. Still images are representative of the cells quantitated in panel D. Scale bars: 10 μm. D) Transiently transfected and stably transduced Jurkat T cells expressing the indicated constructs were stimulated on anti-CD3 and rhVCAM1 and imaged live or after fixation. NEMO clusters were analyzed as in . Data are presented as the mean ± SEM, based on the number of cells analyzed. Numbers in parentheses indicate the number of cells analyzed per condition; numbers in brackets indicate the number of independent experiments from which these cells were derived. Statistical differences among corresponding classes were determined using Student’s T-test: p < 0.05, *; p < 0.01, **; p < 0.001, ***.

Article Snippet: The Jurkat human leukemic T cell line E6.1 (TIB-152; RRID:CVCL_0367) and the C305 hybridoma (CRL-2424; RRID:CVCL_K130) were obtained from ATCC (Manassas, VA).

Techniques: Expressing, Transfection, Flow Cytometry, Stable Transfection, Construct, Derivative Assay

Journal: bioRxiv

Article Title: Polyubiquitin-dependent recruitment of NEMO/IKKγ into T cell receptor signaling microclusters

doi: 10.1101/617126

Figure Lengend Snippet: A) Diagram of NEMO point mutations used in subsequent experiments. B) The functionality of mYFP.NEMO chimeras bearing mutations that impair poly-ubiquitin binding was assessed as in . Data are presented as mean ± SD of 3 technical replicates and are representative of 3-5 experiments. C) Jurkat T cells stably expressing wild-type or mutant mYFP.NEMO chimeras were stimulated on anti-CD3 and rhVCAM1-coated substrates and imaged continuously for at least 5 minutes. Still images are representative of the cells quantitated in panel D. Kymographs were derived from the sub-regions boxed in red. Scale bars: 10 μm. D) Jurkat T cells expressing the indicated constructs were stimulated on anti-CD3 and rhVCAM1. NEMO clusters were imaged, analyzed, and presented as in . E) Primary human T cell blasts were transfected with vectors encoding either a wild-type or a Y301S mutant mYFP.NEMO chimera and a ZAP-70.TRT chimera. Transfected cells were stimulated on anti-CD3, rhVCAM1, and anti-CD28, fixed after 5 minutes, and imaged as above. Images are representative of 4 experiments for NEMO.WT and 2 experiments for NEMO.Y301S. Scale bars: 10 μm.

Article Snippet: The Jurkat human leukemic T cell line E6.1 (TIB-152; RRID:CVCL_0367) and the C305 hybridoma (CRL-2424; RRID:CVCL_K130) were obtained from ATCC (Manassas, VA).

Techniques: Ubiquitin Proteomics, Binding Assay, Stable Transfection, Expressing, Mutagenesis, Derivative Assay, Construct, Transfection

Journal: bioRxiv

Article Title: Polyubiquitin-dependent recruitment of NEMO/IKKγ into T cell receptor signaling microclusters

doi: 10.1101/617126

Figure Lengend Snippet: A) Lck-deficient J.CaM1.6 Jurkat cells were transiently transfected with vectors encoding either mYFP.NEMO alone (left) or mYFP.NEMO and wild-type Lck.Myc.TRT (right), stimulated on substrates coated with anti-CD3 and rhVCAM1, and fixed after 5 minutes. Still images are representative of the cells examined in panel B. B) Jurkat T cells stably expressing wild-type mYFP.NEMO were pre-incubated with 10μM PP2 or a corresponding volume of DMSO for 10 minutes. Cells were stimulated on surfaces coated with anti-CD3 and rhVCAM1 in the presence of the corresponding compounds. Images were acquired continuously for five minutes, beginning two and ten minutes after the addition of cells. Still images derived from the later series are representative of the cells quantitated in panel C. Kymographs derived from both acquisitions are shown. The later kymograph is derived from the region boxed in red. C) Jurkat T cells stably expressing wild-type mYFP.NEMO were pre-treated with 10μM PP2, 100μM piceatannol, or independent DMSO controls, and stimulated on anti-CD3 and rhVCAM1. NEMO clusters were imaged and analyzed as in . D) ZAP-70-deficient P116 Jurkat T cells were transiently transfected with vectors encoding mYFP.NEMO and either a wild-type (WT) or a kinase-dead (K369R, KR) ZAP-70.mCFP chimera. Cells were stimulated on substrates coated with anti-CD3, rhVCAM1, and anti-CD28 and imaged continuously for five minutes. Still images are shown above. Kymographs are derived from the regions boxed in white. Images are representative of two experiments. Scale bars: 10 μm (stills); 5 μm × 60 seconds (kymographs). See for NEMO cluster quantitation in J.CaM1.6 and P116 cells.

Article Snippet: The Jurkat human leukemic T cell line E6.1 (TIB-152; RRID:CVCL_0367) and the C305 hybridoma (CRL-2424; RRID:CVCL_K130) were obtained from ATCC (Manassas, VA).

Techniques: Transfection, Stable Transfection, Expressing, Incubation, Derivative Assay, Quantitation Assay

Journal: bioRxiv

Article Title: Polyubiquitin-dependent recruitment of NEMO/IKKγ into T cell receptor signaling microclusters

doi: 10.1101/617126

Figure Lengend Snippet: A) Wild-type Jurkat T cells and mutant lines lacking LAT (J.CaM2.5), SLP-76 (J14) or Carma1 (JPM50.6) were stably transduced with wild-type mYFP.NEMO, stimulated on anti-CD3 and rhVCAM1, and fixed after five minutes. Still images are representative of the cells examined in panel B. B) Mutant cell lines were prepared and stimulated as in A. NEMO clusters were imaged and analyzed as in . C-D) JPM50.6 Jurkat T cells were transiently transfected with vectors expressing fluorescent chimeras of NEMO and Zap70, and then stimulated, fixed, and imaged as in A. Representative still images and kymographs are shown (n=9 cells in 2 experiments). C) JPM50.6 cells were transfected with vectors expressing mYFP.NEMO and Zap70.mRFP1. Images were acquired continuously for five minutes, beginning two or 20 minutes after the addition of cells. Still images acquired early in each series are shown at left, with kymographs derived from the boxed regions at right (n=4 cells in 2 experiments for each condition). Scale bars: 10 μm (stills); 5 μm × 60 seconds (kymographs).

Article Snippet: The Jurkat human leukemic T cell line E6.1 (TIB-152; RRID:CVCL_0367) and the C305 hybridoma (CRL-2424; RRID:CVCL_K130) were obtained from ATCC (Manassas, VA).

Techniques: Mutagenesis, Stable Transfection, Transduction, Transfection, Expressing, Derivative Assay

Journal: Oncotarget

Article Title: PD-L1 checkpoint blockade delivered by retroviral replicating vector confers anti-tumor efficacy in murine tumor models

doi: 10.18632/oncotarget.26785

Figure Lengend Snippet: Mixtures of SEE loaded, SEE + anti-human PD-L1 (10 µg/mL) or SEE + purified scFv-PD-L1 (10 µg/mL) Raji B cells or Raji B cells overexpressing human PD-L1 (with rested Jurkat T cells overexpressing human PD-1) were co-cultured in RPMI-1640 medium containing 10% human AB serum for stimulation for 20–22 hours followed by IL-2 measurement by ELISA.

Article Snippet: Human leukemic T cell line Jurkat Clone E6-1 (ATCC, TIB-152) and human Burkitt’s B lymphoma cell line Raji (ATCC, CCL86) were cultured in complete RPMI-1640 medium.

Techniques: Purification, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Molecular oral microbiology

Article Title: Lymphoid susceptibility to the Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is dependent upon baseline levels of the signaling lipid, phosphatidylinositol-3,4,5-triphosphate

doi: 10.1111/omi.12127

Figure Lengend Snippet: Comparison of the effects of Cdt on toxicity in Jurkat and HUT78 cells. Jurkat cells were treated with medium alone (panel A) or Cdt (2.0 pg/ml) for 16–20 hr and cell cycle distribution determined following staining with propidium iodide (PI) and analysis by flow cytometry. Panels C and D show HUT78 cells in the presence of medium alone or Cdt, respectively. In addition to cell cycle, the effects of Cdt on apoptosis was assessed using the TUNEL assay; Panels E and F show the results from Jurkat cells in the presence of medium and Cdt (25 pg/ml), respectively, 48 hr after exposure to toxin. Results with HUT78 cells are shown in panels G (medium only) and H (Cdt). Numbers in each panel represent the percentage of G2 cells or apoptotic cells. Panels I and J show the aggregate results from three experiments each performed in duplicate; *indicate P<0.05 when compared to control Jurkat cells and ** indicates P<0.05 when compared to Cdt-treated Jurkat cells.

Article Snippet: Cell culture and cell cycle analysis The human leukemic T cell lines, Jurkat (E6-1) and the T-lymphoblastoid cell line CCRF-CEM, were maintained in RPMI-1640 supplemented with 10% FCS, 2 mM glutamine, 10 mM HEPES, 100 U/ml penicillin and 100 μg/ml streptomycin.

Techniques: Comparison, Staining, Flow Cytometry, TUNEL Assay, Control

Journal: Molecular oral microbiology

Article Title: Lymphoid susceptibility to the Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is dependent upon baseline levels of the signaling lipid, phosphatidylinositol-3,4,5-triphosphate

doi: 10.1111/omi.12127

Figure Lengend Snippet: Baseline analysis of components of the PI-3K signaling pathway. Panel A shows a representative Western blot for pAkt, Akt, pGSK3β (pGSK), GSK3β (GSK) and GAPDH for Jurkat cells, CCRM-CEM cells and HUT78 cells. Panel B shows the aggregate results for three experiments; results are expressed as a percentage of Jurkat cell density. Panel C shows the results of PIP3 levels and represent the mean SEM. *indicate P<0.05 when compared to control Jurkat cells

Article Snippet: Cell culture and cell cycle analysis The human leukemic T cell lines, Jurkat (E6-1) and the T-lymphoblastoid cell line CCRF-CEM, were maintained in RPMI-1640 supplemented with 10% FCS, 2 mM glutamine, 10 mM HEPES, 100 U/ml penicillin and 100 μg/ml streptomycin.

Techniques: Western Blot, Control

Journal: Molecular oral microbiology

Article Title: Lymphoid susceptibility to the Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is dependent upon baseline levels of the signaling lipid, phosphatidylinositol-3,4,5-triphosphate

doi: 10.1111/omi.12127

Figure Lengend Snippet: Analysis of Jurkat cells reconstituted with PTEN. Panel A shows the baseline PIP3 levels were measured in Jurkat cells reconstituted with PTEN: controls (Con18) and pten transfection (PIJ-12); these cells were assessed under non-induced and induced conditions. Values are pmol PIP3/106 cells and represent the mean ± SEM of three replicate experiments; *indicate P<0.05 when compared to uninduced cells treated with Cdt. Panel B shows the effect of Cdt holotoxin on PIJ-12 and Con 18 cell lines which were treated with doxycycline as well as cells maintained under non-induced conditions. The percentage of G2 cells is plotted versus Cdt concentration. The mean ± SEM is shown for three experiments; *indicate P<0.05 when compared touninduced cells treated with Cdt.

Article Snippet: Cell culture and cell cycle analysis The human leukemic T cell lines, Jurkat (E6-1) and the T-lymphoblastoid cell line CCRF-CEM, were maintained in RPMI-1640 supplemented with 10% FCS, 2 mM glutamine, 10 mM HEPES, 100 U/ml penicillin and 100 μg/ml streptomycin.

Techniques: Transfection, Concentration Assay

Journal: Molecular oral microbiology

Article Title: Lymphoid susceptibility to the Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is dependent upon baseline levels of the signaling lipid, phosphatidylinositol-3,4,5-triphosphate

doi: 10.1111/omi.12127

Figure Lengend Snippet: Flow cytometric analysis of the binding of Cdt holotoxin and internalization of CdtB in Jurkat cells and HUT78 cells. Jurkat cells (panels A and B) and HUT78 cells (panels C and D) were exposed to medium (panels A and C) or Cdt (panels B and D). Cells were then stained for surface association of Cdt holotoxin (anti-CdtC mAb) or internalization of CdtB (anti-CdtB mAb). The mean channel fluorescence (MCF) is indicated in each panel for both control cells not exposed to Cdt (grey curve) and toxin treated cells (black line). The mean MCF ± SEM for three experiments is shown in panel E; *indicate P<0.05 when compared to control cells.

Article Snippet: Cell culture and cell cycle analysis The human leukemic T cell lines, Jurkat (E6-1) and the T-lymphoblastoid cell line CCRF-CEM, were maintained in RPMI-1640 supplemented with 10% FCS, 2 mM glutamine, 10 mM HEPES, 100 U/ml penicillin and 100 μg/ml streptomycin.

Techniques: Binding Assay, Staining, Fluorescence, Control

Journal: PLoS ONE

Article Title: Phosphorylation of SET Protein at Ser171 by Protein Kinase D2 Diminishes Its Inhibitory Effect on Protein Phosphatase 2A

doi: 10.1371/journal.pone.0051242

Figure Lengend Snippet: Activated PKD2 was immunoprecipitated from lysate of Jurkat cells stimulated with anti-CD3ε antibody. The heat-inactivated (50°C, 5 min) Jurkat nuclear extract was used as a substrate for PKD2 and analyzed by 2D-gel electrophoresis. (A) Activated PKD2 was incubated with the nuclear extracts in the presence of [γ- 32 P]-ATP and Gö6983 (an irrelevant PKC inhibitor) and the radioactivity of 2D-electrophregram was detected. (B) Activated PKD2 was incubated with the nuclear extracts in the presence of [γ- 32 P]-ATP and Gö6976 (PKD2 inhibitor) and the radioactivity of 2D-electrophoregram was detected. (C) The proteins in the Jurkat nuclear extract treated with non-radioactive ATP and PKD2 were stained with syproruby. Inset; the enlarged view of the boxed area. A protein spot indicated by the asterisks, which overlaps to the radioactive spots indicated in A or in B was analyzed by mass spectrometry. (D) One of the MS/MS spectra assigned to be SET protein is shown (m/z = 604, 110 VEVTEFEDIK 119 ).

Article Snippet: Human leukemic T cell line Jurkat (E6-1) was obtained from American Type Culture Collection (Manassas, VA).

Techniques: Immunoprecipitation, Two-Dimensional Gel Electrophoresis, Electrophoresis, Incubation, Radioactivity, Staining, Mass Spectrometry, Tandem Mass Spectroscopy

Journal: PLoS ONE

Article Title: Phosphorylation of SET Protein at Ser171 by Protein Kinase D2 Diminishes Its Inhibitory Effect on Protein Phosphatase 2A

doi: 10.1371/journal.pone.0051242

Figure Lengend Snippet: (A) After Jurkat cells expressing GFP-SET were stimulated by PHA (2 µg/ml) with or without Gö6976 (3 µM), the phosphorylated GFP-tagged SET was collected by PhosphoProtein Purification kit and separated by SDS-PAGE. The phosphoprotein was transferred onto the PVDF membrane and quantified by ECL using anti-GFP antibody and the secondary antibody. The typical blot was shown in the lower-right panel. The luminescence of samples from cells without both stimulation and the inhibitor was assigned to be 1. The results were expressed as mean +SD for three independent experiments. *, p<0.01; **, p<0.05. As shown in the upper-right panel, phosphorylated GFP was not detected from the eluate of the PHA stimulated Jurkat cells expressing GFP only. (B) Jurkat cells expressing GFP-SET treated with control (ctrl) siRNA1 had no effect on the PKD2 expression, while siRNA2 markedly reduced the PKD2 expression (upper-right panel). The typical blot was shown in the lower-right panel. The luminescence of the samples from non-stimulated and siRNA1-treated cells was assigned to be 1. The results were expressed as mean +SD for three independent experiments. ***, p<0.05; ****, p<0.05, as determined by Student's t test.

Article Snippet: Human leukemic T cell line Jurkat (E6-1) was obtained from American Type Culture Collection (Manassas, VA).

Techniques: Expressing, Purification, SDS Page, Membrane, Control

Journal: PLoS ONE

Article Title: Phosphorylation of SET Protein at Ser171 by Protein Kinase D2 Diminishes Its Inhibitory Effect on Protein Phosphatase 2A

doi: 10.1371/journal.pone.0051242

Figure Lengend Snippet: The Jurkat cells expressing GFP-SET-S171A were stimulated and phosphorylation status of GFP-SET-S171A was investigated by the same method as described in . The results were indicated as mean +SD for three independent experiments. There was no significant up- or down-regulation of recovered phospho-GFP-SET-S171A by PHA stimulation with or without PKD2 inhibitor, suggesting that Ser171 of SET is the major target site of TCR-activated PKD2.

Article Snippet: Human leukemic T cell line Jurkat (E6-1) was obtained from American Type Culture Collection (Manassas, VA).

Techniques: Expressing, Phospho-proteomics

Journal: PLoS ONE

Article Title: Phosphorylation of SET Protein at Ser171 by Protein Kinase D2 Diminishes Its Inhibitory Effect on Protein Phosphatase 2A

doi: 10.1371/journal.pone.0051242

Figure Lengend Snippet: (A) Human CD4 + T cell clone was stimulated by L cells expressing its cognate ligands in the presence (closed circles) or absence (open circlse) of PKD2 inhibitor Gö6976 (3 µM). (B) Jurkat cells treated with siRNA were stimulated by anti-CD3ε antibody and anti-mouse IgG antibody. Treatment with siRNA2 (closed circles) significantly reduced the PKD2 expression, while siRNA1 (open circles) had almost no effect and hence used as negative controls (right panel). (C) Normal Jurkat cells (open circles) and Jurkat cells expressing GFP-SET-S171A (closed circles) were stimulated by anti-CD3ε antibody and anti-mouse IgG antibody. The latter cells expressed 4.5-fold higher GFP-SET-S171A than the endogenous SET as judged by the intensity of the bands using anti-SET antibody on the Western blot (upper box). Cell lysate were subjected to SDS-PAGE followed by western blot using anti-PP2A phospho-Tyr307 antibody and horseradish peroxidase-conjugated second antibody. The amount of each band was quantified by using ECL and LAS-4000. The same membrane was stripped and reprobed with the anti-PP2A C-subunit antibody to monitor relative abundance of total PP2A protein. Values represent the means ±SD from three independent experiments.

Article Snippet: Human leukemic T cell line Jurkat (E6-1) was obtained from American Type Culture Collection (Manassas, VA).

Techniques: Expressing, Western Blot, SDS Page, Membrane

Journal: PLoS ONE

Article Title: Phosphorylation of SET Protein at Ser171 by Protein Kinase D2 Diminishes Its Inhibitory Effect on Protein Phosphatase 2A

doi: 10.1371/journal.pone.0051242

Figure Lengend Snippet: Normal and GFP-SET-S171A-overexpressing Jurkat cells were stimulated by anti-CD3e antibody and anti-mouse IgG antibody for 7 min were lyzed and subjected to SDS-PAGE followed by the western blot analyses. The phospho-ERK (p-ERK) was first detected and quantified and after stripping off the antibodies, ERK1 was detected and quantified to monitor p-ERK/ERK1 ratio. The p-ERK/ERK1 ratio of Jurkat cell was assigned to be 1. The results were indicated as mean +SD for three independent experiments. *, p<0.01, as determined by Student's t test.

Article Snippet: Human leukemic T cell line Jurkat (E6-1) was obtained from American Type Culture Collection (Manassas, VA).

Techniques: SDS Page, Western Blot, Stripping Membranes