Journal:

Article Title: Epitope-specific crosslinking of CD45 down-regulates membrane-associated tyrosine phosphatase activity and triggers early signalling events in human activated T cells

doi: 10.1111/j.1365-2567.2004.01986.x

Figure Lengend Snippet: TNF-α gene expression requires PTK and PKC activity. Primary activated T cells were preincubated for 30 min with PTK inhibitors genistein (a), tyrphostin (b), herbimycin (c), or PP2 (d), or with the PKC inhibitor calphostin C (e), then simulated for 2 hr with anti-CD45 mAb 4B2 crosslinked on plastic. Similar results were obtained with cells stimulated with mAb 9.4. Alternatively, T cells preincubated with tyrphostin were stimulated with PMA (20 ng/ml) and Ionomycin (1 µm) (f). Results are expressed as TNF-α mRNA fold induction over unstimulated T cells (representative sample in (a)). [32P]-labelled PCR products quantified using an Instant Imager™ are presented in boxes (TNF-α transcripts: upper signal, internal standard transcripts: lower band).

Article Snippet: Primary activated human T cells or Jurkat T cells (1 × 10 7 ) were first preincubated with soluble anti-CD45 mAb (20 μg/ml) for 3 min, washed in culture medium, and subsequently crosslinked with rabbit or goat affinity-purified anti-mouse IgG (20 μg/ml; Cappel, Durham, UK).

Techniques: Gene Expression, Activity Assay

Journal:

Article Title: Epitope-specific crosslinking of CD45 down-regulates membrane-associated tyrosine phosphatase activity and triggers early signalling events in human activated T cells

doi: 10.1111/j.1365-2567.2004.01986.x

Figure Lengend Snippet: Upstream PTK p56lck, ZAP-70 and p72syk, adaptor protein LAT and exchange factor Vav are phosphorylated upon CD45 engagement. Jurkat T cells were stimulated with soluble (S) or crosslinked (XL) anti-CD45 mAbs 9.4 (or 4B2, data not shown) or UCHL1 (20 µg/ml) (or 10G10, data not shown), or left unstimulated for increasing periods of time. Whole cell lysates were immunoprecipitated with anti-p56lck, anti-ZAP-70, anti-p72syk, anti-LAT and anti-Vav antibodies. (a, b) Following separation on 6·5–10% polyacrylamide gradient gels in SDS and transfer to PVDF membranes, blots were incubated with antiphosphotyrosine mAb and revealed by chemiluminescence. (c, d) Blots were stripped prior to incubation with specific anti-p56lck, anti-ZAP-70, antip72syk, anti-CD3ζ, anti-LAT and anti-Vav antibodies.

Article Snippet: Primary activated human T cells or Jurkat T cells (1 × 10 7 ) were first preincubated with soluble anti-CD45 mAb (20 μg/ml) for 3 min, washed in culture medium, and subsequently crosslinked with rabbit or goat affinity-purified anti-mouse IgG (20 μg/ml; Cappel, Durham, UK).

Techniques: Immunoprecipitation, Incubation

Journal:

Article Title: Epitope-specific crosslinking of CD45 down-regulates membrane-associated tyrosine phosphatase activity and triggers early signalling events in human activated T cells

doi: 10.1111/j.1365-2567.2004.01986.x

Figure Lengend Snippet: Tyrosine phosphorylation of PTK ZAP-70 and p72syk, adaptor protein LAT and p23-ζ induced by CD45 engagement is dependent on p56lck kinase activity. Jurkat T cells were preincubated or not with PP2 (10 or 100 µm) for 30 min, and were then stimulated with soluble (S) or crosslinked (XL) anti-CD45 mAbs 9.4 (20 µg/ml) (or 4B2, data not shown) for 15 min, or left unstimulated. Whole cell lysates were immunoprecipitated with specific anti-p72syk, anti-ZAP-70, anti-p56lck, anti-LAT and anti-CD3-ζ antibodies. (a) Following separation on 6·5–10% polyacrylamide gradient gels in SDS and transfer to PVDF membranes, blots were incubated with antiphosphotyrosine mAb and revealed by chemiluminescence. (b) Blots were stripped prior to incubation with antip72syk, anti-ZAP-70, anti-p56lck, anti-LAT and anti-CD3-ζ antibodies.

Article Snippet: Primary activated human T cells or Jurkat T cells (1 × 10 7 ) were first preincubated with soluble anti-CD45 mAb (20 μg/ml) for 3 min, washed in culture medium, and subsequently crosslinked with rabbit or goat affinity-purified anti-mouse IgG (20 μg/ml; Cappel, Durham, UK).

Techniques: Phospho-proteomics, Activity Assay, Immunoprecipitation, Incubation

Journal:

Article Title: Epitope-specific crosslinking of CD45 down-regulates membrane-associated tyrosine phosphatase activity and triggers early signalling events in human activated T cells

doi: 10.1111/j.1365-2567.2004.01986.x

Figure Lengend Snippet: Membrane-associated PTPase activity is down-regulated by CD45 crosslinking in an epitope-dependent manner. T cells (106; a, primary activated T cells; b, Jurkat T cells) stimulated for 30 min with soluble or crosslinked anti-CD45 mAbs 9.4, 4B2, 10G10 or UCHL1, or with control anti-MHC class II mAb 3B12 (each at 20 µg/ml) were disrupted in hypotonic buffer, and the membrane cytoskelettal fraction isolated by ultracentrifugation. PTPase activity was measured using a colorimetric assay with pNPP as substrate. Activity from samples treated with crosslinked mAb was expressed as percent inhibition of that obtained from samples treated with soluble mAb. Data represents the mean ± SD from three independent experiments.

Article Snippet: Primary activated human T cells or Jurkat T cells (1 × 10 7 ) were first preincubated with soluble anti-CD45 mAb (20 μg/ml) for 3 min, washed in culture medium, and subsequently crosslinked with rabbit or goat affinity-purified anti-mouse IgG (20 μg/ml; Cappel, Durham, UK).

Techniques: Membrane, Activity Assay, Control, Isolation, Colorimetric Assay, Inhibition

Journal:

Article Title: Epitope-specific crosslinking of CD45 down-regulates membrane-associated tyrosine phosphatase activity and triggers early signalling events in human activated T cells

doi: 10.1111/j.1365-2567.2004.01986.x

Figure Lengend Snippet: Broad spectrum PTPase inhibitors block TNF-α gene expression. Primary activated T cells (3 × 106) were preincubated for 30 min (a) with PTPase inhibitors PAO, or (b) with sodium pervanadate (Na3VO4/H2O2), then stimulated for 2 hr with anti-CD45 mAb 4B2 (20 µg/ml)crosslinked on plastic. (c) Primary activated T cells were incubated (30 min) with the serine/threonine phosphatase inhibitor okadaic acid prior to stimulation with anti-CD45 mAb 4B2 as for (a) and (b), or left unstimulated (unstim.). Total RNA was isolated and analysed by RT-PCR using TNF-α-specific primers. Results are expressed as fold induction over unstimulated T cells. Similar results were obtained with cells stimulated with mAb 9.4.

Article Snippet: Primary activated human T cells or Jurkat T cells (1 × 10 7 ) were first preincubated with soluble anti-CD45 mAb (20 μg/ml) for 3 min, washed in culture medium, and subsequently crosslinked with rabbit or goat affinity-purified anti-mouse IgG (20 μg/ml; Cappel, Durham, UK).

Techniques: Blocking Assay, Gene Expression, Incubation, Isolation, Reverse Transcription Polymerase Chain Reaction

Journal:

Article Title: Epitope-specific crosslinking of CD45 down-regulates membrane-associated tyrosine phosphatase activity and triggers early signalling events in human activated T cells

doi: 10.1111/j.1365-2567.2004.01986.x

Figure Lengend Snippet: PTK activity is not enhanced in response to CD45 engagement. Following stimulation of Jurkat T cells with soluble (S) or crosslinked (XL) anti-CD45 mAb 9.4, 4B2, 10G10 or UCHL1 for 15 min at 37°, p56lck, ZAP-70 or p72syk (data not shown) were immunoprecipitated with specific antibodies. (a) Equivalent amounts of p56lck (as evaluated by immunoblotting using an anti-p56lck mAb) were assayed (b) for kinase autophosphorylation and for phosphorylation of rabbit enolase (10 min) (incorporation of [32P]-phosphate into rabbit enolase revealed by autoradiography). (c, d) Assessment of ZAP-70 kinase activity was conducted similarly.

Article Snippet: Primary activated human T cells or Jurkat T cells (1 × 10 7 ) were first preincubated with soluble anti-CD45 mAb (20 μg/ml) for 3 min, washed in culture medium, and subsequently crosslinked with rabbit or goat affinity-purified anti-mouse IgG (20 μg/ml; Cappel, Durham, UK).

Techniques: Activity Assay, Immunoprecipitation, Western Blot, Phospho-proteomics, Autoradiography

Journal:

Article Title: Epitope-specific crosslinking of CD45 down-regulates membrane-associated tyrosine phosphatase activity and triggers early signalling events in human activated T cells

doi: 10.1111/j.1365-2567.2004.01986.x

Figure Lengend Snippet: Epitope-specific CD45 crosslinking induces phosphoinositide turnover and intracellular calcium fluxes. (a) Jurkat T cells were loaded with myo-2-[3H]-inositol and stimulated for 35 min with increasing concentrations of crosslinked mAb 9.4 (or mAb 4B2, data not shown), or left unstimulated. Total [3H]-phosphoinositide generation was expressed in counts per minute (mean of triplicates ± SD). (b), Jurkat T cells loaded with myo-2-[3H]-inositol were stimulated for 35 min with a panel of crosslinked anti-CD45 mAbs (10G10, UCHL1, 9.4: 20 µg/ml), or control antibodies (UPC10, 20 µg/ml; anti-CD3 mAb OKT3, 1 µg/ml). Total [3H]-phosphoinositide generation was expressed in counts per min (mean of triplicates ± SD). (c) Jurkat T cells loaded with Indo-1 were stimulated for 3 min with soluble (S) anti-CD45 mAb 4B2, 9.4 or UCHL1, then crosslinked (XL) with goat anti-mouse antibodies. Results are expressed as FL2 fluorescence ratios over time. Intracellular Ca2+ rise induced by mAb 9.4 crosslinking was inhibited by pretreatment with herbimycin A 10 µg/ml for 30 min.

Article Snippet: Primary activated human T cells or Jurkat T cells (1 × 10 7 ) were first preincubated with soluble anti-CD45 mAb (20 μg/ml) for 3 min, washed in culture medium, and subsequently crosslinked with rabbit or goat affinity-purified anti-mouse IgG (20 μg/ml; Cappel, Durham, UK).

Techniques: Control, Fluorescence