src family kinase–specific inhibitor pp1  (Millipore)

Journal:

Article Title: Transient alteration of T cell fine specificity by a strong primary stimulus correlates with T cell receptor down-regulation

doi:

Figure Lengend Snippet: Inhibition of protein tyrosine kinase activity blocks TCR down-regulation. P14 TCR transgenic spleen cells (A, B) or P14.G5 CTL clone (C, D) were preincubated for 60 min in the presence of PP1 or genistein. Preincubation with herbimycin A was done overnight with P14 spleen cells or for 60 min with P14.G5 CTL. EL-4 cells pulsed with p33 (1 μM) were then added for 4 h and cells stained for flow cytometry with anti-CD8 and anti-Vα2 antibodies (A, C) or with anti-CD8 and anti-CD69 antibodies (B, D). Mean fluorescence intensities are expressed as % or MFI of control in the absence of peptide.

Article Snippet: 4.13 Inhibition of src family kinase activity P14.G5 CTL (6×10 4 /well) or P14 spleen cells (5×10 5 /well) were preincubated for 60 min with the src family kinase-specific inhibitor PP1 (10 μM and 50 μM, respectively) (Bio-mol, Plymouth Meeting, PA) [ 28 ].

Techniques: Inhibition, Activity Assay, Transgenic Assay, Staining, Flow Cytometry, Fluorescence, Control

Journal: The Journal of Experimental Medicine

Article Title: Phosphoprotein Associated with Glycosphingolipid-Enriched Microdomains (Pag), a Novel Ubiquitously Expressed Transmembrane Adaptor Protein, Binds the Protein Tyrosine Kinase Csk and Is Involved in Regulation of T Cell Activation

doi:

Figure Lengend Snippet: Posttranslational modifications of PAG. (A) Lysates of pervanadate-treated (+) or untreated (−) Jurkat cells were analyzed by anti-PAG Western blotting. (B) COS cells were transiently transfected with the depicted cDNA constructs. Lysates corresponding to 10% of the transfectants were analyzed by P-Tyr Western blotting (top left). The blot was stripped, and expression of the individual constructs was assessed using FLAG (FLAG-PAG and FLAG-Syk), Lck, Fyn, and MYC (MYC-ZAP70) Abs, respectively (right). The remaining 90% of the lysates were subjected to anti-FLAG immunoprecipitation and analyzed by P-Tyr Western blotting (bottom left). Arrowheads indicate the positions of phosphorylated PAG. (C) Anti-PAG (left) and anti–P-Tyr (right) Western blots of total cell lysates of wild-type Jurkat cells, the Lck-negative mutant J.CaM1.6, and the ZAP70/Syk-negative mutant P116. (D) Lysates of Jurkat cells treated for 1 min with 10 μM inhibitor of Src family PTKs PP1 (left) or untreated controls (Ctr; right) were analyzed by anti–P-Tyr blot. The blots were stripped and reincubated with mAb to PAG (bottom).

Article Snippet: The Src family kinase–specific inhibitor PP1 (Calbiochem; provided by Dr. P. Dráber, Institute of Molecular Genetics, Prague, Czech Republic; reference 45) was used at 10 μg/ml.

Techniques: Western Blot, Transfection, Construct, Expressing, Immunoprecipitation, Mutagenesis

Journal: The Journal of Experimental Medicine

Article Title: Phosphoprotein Associated with Glycosphingolipid-Enriched Microdomains (Pag), a Novel Ubiquitously Expressed Transmembrane Adaptor Protein, Binds the Protein Tyrosine Kinase Csk and Is Involved in Regulation of T Cell Activation

doi:

Figure Lengend Snippet: Fyn and and Csk associate with PAG. (A) In vitro pull-down assay. Lysates of untreated (–) or pervanadate (PV)-treated (+) Jurkat cells were subjected to precipitation using the depicted recombinant GST–SH2 domain fusion proteins. Subsequently, anti-PAG or anti-TRIM Western blotting was performed. (B) Resting peripheral blood T cells (3 × 10 7 ) were left untreated or were treated with the Src family kinase inhibitor PP1 (10 μM) for 2 min. Subsequently, PAG immunoprecipitates obtained from laurylmaltoside lysates were subjected to sequential anti–P-Tyr, anti-Fyn, and anti-Csk immunoblotting. (C) Untreated Raji, Jurkat, or peripheral blood α/β T cells were solubilized by 1% laurylmaltoside followed by PAG or control immunoprecipitation and Csk Western blotting. L, original lysates. (D) The blots shown in B (corresponding to both total lysates [bottom] and anti-FLAG immunoprecipitates [middle]) were stripped and incubated with a polyclonal antiserum directed against Csk. (E) COS cells (expressing endogenous Csk) were transfected with cDNA constructs encoding tyrosine mutants of FLAG-PAG and the PTK Fyn. Anti-FLAG immunoprecipitates were analyzed by Western blotting for the presence of Csk and Fyn. The numbers at the top identify the Tyr residues mutated to Phe. The expression levels of Fyn and Csk in total lysates were determined in parallel (bottom strips). None of the investigated PAG mutants showed any gross alterations of the overall level of tyrosine phosphorylation (not shown).

Article Snippet: The Src family kinase–specific inhibitor PP1 (Calbiochem; provided by Dr. P. Dráber, Institute of Molecular Genetics, Prague, Czech Republic; reference 45) was used at 10 μg/ml.

Techniques: In Vitro, Pull Down Assay, Recombinant, Western Blot, Control, Immunoprecipitation, Incubation, Expressing, Transfection, Construct, Phospho-proteomics