Journal:

Article Title: Recruitment of the Class II Phosphoinositide 3-Kinase C2? to the Epidermal Growth Factor Receptor: Role of Grb2

doi: 10.1128/MCB.21.19.6660-6667.2001

Figure Lengend Snippet: PI3K-C2β(2-298) affinity purifies EGFR, Shc, and Grb2 in a dose-dependent manner. Lysates from EGF-stimulated A431 cells were incubated with various additions of recombinant GST–PI3K-C2β(2-298) protein as shown. After 4 h at 4°C the fusion and associated proteins were isolated by centrifugation and were washed. They were then extracted, fractionated by SDS-PAGE, and Western blotted with antiphosphotyrosine (upper panel), anti-Shc (middle panel), or anti-Grb2 (lower panel). PY, phosphotyrosine.

Article Snippet: Confluent and quiescent cultures of human renal epithelial cells (HKC-8) and breast cancer cells (HTM3551, T47D, and SKBr3) were incubated with (+) or without (−) EGF (100 nM) for 10 min. Lysates were prepared to which anti-Grb2 antibody (Santa Cruz C-23) was added for 4 h at 4°C.

Techniques: Incubation, Recombinant, Isolation, Centrifugation, SDS Page, Western Blot

Journal:

Article Title: Recruitment of the Class II Phosphoinositide 3-Kinase C2? to the Epidermal Growth Factor Receptor: Role of Grb2

doi: 10.1128/MCB.21.19.6660-6667.2001

Figure Lengend Snippet: N-terminal fragments of PI3K-C2β establish that two proline-rich motifs are required for association with EGFR. A series of GST fusion proteins representing residues 2 to 130, 2 to 143, 2 to 157, 2 to 255, and 2 to 298 were incubated for 4 h at 4°C with lysates of A431 cells that had been treated in the absence (−) or presence (+) of EGF (100 nM) for 10 min. For control, EGFR was also immunoprecipitated from these lysates (Ab1; Oncogene Science). Each fusion and associated protein was isolated using glutathione-Sepharose beads, washed, fractionated by SDS-PAGE, and Western blotted with antiphosphotyrosine antibody to visualize the activated EGFR (upper panel) and anti-Grb2 antibody (lower panel). PY, phosphotyrosine.

Article Snippet: Confluent and quiescent cultures of human renal epithelial cells (HKC-8) and breast cancer cells (HTM3551, T47D, and SKBr3) were incubated with (+) or without (−) EGF (100 nM) for 10 min. Lysates were prepared to which anti-Grb2 antibody (Santa Cruz C-23) was added for 4 h at 4°C.

Techniques: Incubation, Immunoprecipitation, Isolation, SDS Page, Western Blot

Journal:

Article Title: Recruitment of the Class II Phosphoinositide 3-Kinase C2? to the Epidermal Growth Factor Receptor: Role of Grb2

doi: 10.1128/MCB.21.19.6660-6667.2001

Figure Lengend Snippet: Recombinant Grb2 and PI3K-C2β associate in vitro. Grb2-GST fusion protein (1 μg) was added to Triton lysis buffer (500 μl) in the absence or presence of EE epitope-tagged PI3K-C2β (1 μg) and either glutathione-Sepharose beads or anti-EE tag antibody (Ab) and protein A-Sepharose. After incubation at 4°C for 4 h, beads were isolated by centrifugation and washed. Associated proteins were extracted, fractionated by SDS-PAGE, and Western blotted with either anti-PI3K-C2β antibody (upper panel) or anti-Grb2 antibody (lower panel) and visualized with ECL.

Article Snippet: Confluent and quiescent cultures of human renal epithelial cells (HKC-8) and breast cancer cells (HTM3551, T47D, and SKBr3) were incubated with (+) or without (−) EGF (100 nM) for 10 min. Lysates were prepared to which anti-Grb2 antibody (Santa Cruz C-23) was added for 4 h at 4°C.

Techniques: Recombinant, In Vitro, Lysis, Incubation, Isolation, Centrifugation, SDS Page, Western Blot

Journal:

Article Title: Recruitment of the Class II Phosphoinositide 3-Kinase C2? to the Epidermal Growth Factor Receptor: Role of Grb2

doi: 10.1128/MCB.21.19.6660-6667.2001

Figure Lengend Snippet: Both N-terminal and C-terminal Grb2 SH3 domains (N-SH3 and C-SH3, respectively) bind PI3K-C2β. The N-terminal and C-terminal SH3 domains of Grb2 together with full-length Grb2 adaptor were expressed as GST fusion proteins and visualized by Coomassie blue staining following SDS-PAGE (panel A). The N-terminal PI3K-C2β fragments 2-130, 2-143, 2-157, 2-255, and 2-298 were expressed as GST fusion proteins, purified using glutathione-Sepharose beads, and cleaved with 5 μg of thrombin/ml (2 h, 21°C). Each protein was fractionated by SDS-PAGE and Western blotted with anti-PI3K-C2β antisera to confirm cross-reaction (panel B). Aliquots of each PI3K-C2β N-terminal fragment were incubated (2 h, 4°C) with either GST, full-length Grb2, or N-terminal or C-terminal Grb2 SH3 domain. Each GST fusion was isolated with glutathione-Sepharose beads, washed, and fractionated by SDS-PAGE. The N-terminal fragments of PI3K-C2β were visualized by Western blotting with anti-PI3K-C2β antibody.

Article Snippet: Confluent and quiescent cultures of human renal epithelial cells (HKC-8) and breast cancer cells (HTM3551, T47D, and SKBr3) were incubated with (+) or without (−) EGF (100 nM) for 10 min. Lysates were prepared to which anti-Grb2 antibody (Santa Cruz C-23) was added for 4 h at 4°C.

Techniques: Staining, SDS Page, Purification, Western Blot, Incubation, Isolation

Journal:

Article Title: Recruitment of the Class II Phosphoinositide 3-Kinase C2? to the Epidermal Growth Factor Receptor: Role of Grb2

doi: 10.1128/MCB.21.19.6660-6667.2001

Figure Lengend Snippet: Isolation of PI3K-C2β, SOS, and EGFR in anti-Grb2 immunoprecipitates (ippt). Confluent and quiescent cultures of human renal epithelial cells (HKC-8) and breast cancer cells (HTM3551, T47D, and SKBr3) were incubated with (+) or without (−) EGF (100 nM) for 10 min. Lysates were prepared to which anti-Grb2 antibody (Santa Cruz C-23) was added for 4 h at 4°C. Immune complexes were isolated following addition of protein A-Sepharose and centrifugation. Proteins were extracted, fractionated by SDS-PAGE, and Western blotted with antisera to PI3K-C2β (upper panels), SOS 1/2 (D21; Santa Cruz) (middle panels), and EGFR (1005; Santa Cruz) (lower panels).

Article Snippet: Confluent and quiescent cultures of human renal epithelial cells (HKC-8) and breast cancer cells (HTM3551, T47D, and SKBr3) were incubated with (+) or without (−) EGF (100 nM) for 10 min. Lysates were prepared to which anti-Grb2 antibody (Santa Cruz C-23) was added for 4 h at 4°C.

Techniques: Isolation, Incubation, Centrifugation, SDS Page, Western Blot

Journal:

Article Title: Recruitment of the Class II Phosphoinositide 3-Kinase C2? to the Epidermal Growth Factor Receptor: Role of Grb2

doi: 10.1128/MCB.21.19.6660-6667.2001

Figure Lengend Snippet: Grb2 associates with PI3K-C2β in vivo. Cultures of confluent and quiescent A431, LNCaP, and HEK293 cells that overexpress epitope-tagged PI3K-C2β were incubated in the absence (−) or presence (+) of EGF (100 nM) for 10 min. Lysates were prepared and immunoprecipitated (ippt) with anti-Grb2 antisera for 4 h at 4°C. Immune complexes were isolated with protein A-Sepharose. These were either extracted, fractionated by SDS-PAGE, and Western blotted with anti-PI3K-C2β antisera (upper panels) or used for lipid kinase assay with PtdIns as the substrate and Ca2+ as the divalent cation (lower panels). The activity of recombinant PI3K-C2β is shown as a control, as is the class IA PI3K activity immunoprecipitated from A431 cell lysates using anti-p85α antibody.

Article Snippet: Confluent and quiescent cultures of human renal epithelial cells (HKC-8) and breast cancer cells (HTM3551, T47D, and SKBr3) were incubated with (+) or without (−) EGF (100 nM) for 10 min. Lysates were prepared to which anti-Grb2 antibody (Santa Cruz C-23) was added for 4 h at 4°C.

Techniques: In Vivo, Incubation, Immunoprecipitation, Isolation, SDS Page, Western Blot, Kinase Assay, Activity Assay, Recombinant

Journal:

Article Title: Recruitment of the Class II Phosphoinositide 3-Kinase C2? to the Epidermal Growth Factor Receptor: Role of Grb2

doi: 10.1128/MCB.21.19.6660-6667.2001

Figure Lengend Snippet: Grb2 increases the catalytic activity of PI3K-C2β. Aliquots of recombinant EE-tagged PI3K-C2β (100 ng) were incubated in PI3K assay buffer for 2 h at 4°C with either Grb2-GST fusion protein (100 ng) or GST. After this time, immobilized EGFR previously immunoprecipitated (Ab-1; Oncogene Science) from lysates of quiescent A431 cells and autophosphorylated in vitro or mock control was added. Incubation was continued for a further 1 h. Following addition of kinase buffer, phosphatidylinositol (200 μg/ml) and [γ-32P]ATP, samples (50 μl) were incubated at room temperature for 20 min. Radiolabeled phosphoinositides were extracted and aliquots were fractionated by thin-layer chromatography and visualized by autoradiography. Representative data are shown in the upper panel. Quantification of PtdIns3P was undertaken by scanning densitometry. In the lower panel, data are displayed as means ± standard errors of the means (n = 6). Under the conditions used, all reactions displayed linear kinetics.

Article Snippet: Confluent and quiescent cultures of human renal epithelial cells (HKC-8) and breast cancer cells (HTM3551, T47D, and SKBr3) were incubated with (+) or without (−) EGF (100 nM) for 10 min. Lysates were prepared to which anti-Grb2 antibody (Santa Cruz C-23) was added for 4 h at 4°C.

Techniques: Activity Assay, Recombinant, Incubation, Immunoprecipitation, In Vitro, Thin Layer Chromatography, Autoradiography

Journal:

Article Title: Recruitment of the Class II Phosphoinositide 3-Kinase C2? to the Epidermal Growth Factor Receptor: Role of Grb2

doi: 10.1128/MCB.21.19.6660-6667.2001

Figure Lengend Snippet: Formation of the EGFR-Grb2–PI3K-C2β complex in vitro. Recombinant EE-tagged PI3K-C2β (100 ng) was incubated in lysis buffer for 2 h at 4°C in the absence or presence of Grb2-GST fusion protein (100 ng) or GST. Immobilized EGFR, isolated by immunoprecipitation (Ab1; Oncogene Science) from lysates of confluent and quiescent cultures of A431 cells, was phosphorylated for 30 min at 30°C in protein kinase buffer upon addition of ATP. Either phosphorylated EGFR, nonphosphorylated EGFR, or mock immunoprecipitate was added to the Grb2–PI3K-C2β sample, and the incubation was continued for a further 1 h. Beads containing immobilized receptor and associated proteins were isolated by centrifugation, washed, fractionated by SDS-PAGE, and Western blotted with either anti-EGFR antibody, antiphosphotyrosine, anti-Grb2, or anti-PI3K-C2β antibody. PY, phosphotyrosine.

Article Snippet: Confluent and quiescent cultures of human renal epithelial cells (HKC-8) and breast cancer cells (HTM3551, T47D, and SKBr3) were incubated with (+) or without (−) EGF (100 nM) for 10 min. Lysates were prepared to which anti-Grb2 antibody (Santa Cruz C-23) was added for 4 h at 4°C.

Techniques: In Vitro, Recombinant, Incubation, Lysis, Isolation, Immunoprecipitation, Centrifugation, SDS Page, Western Blot

Journal:

Article Title: Tat protein induces self-perpetuating permissivity for productive HIV-1 infection

doi:

Figure Lengend Snippet: Aberrant activation of lymphocytes by extracellular Tat is associated with G0 to G1 progression but not entry into S phase. (a) Primary lymphocytes remained quiescent after 72 h in culture. (b) Cells activated by extracellular Tat did not enter S phase, unlike (c) cells activated by phytohemagglutinin (PHA-C; Boehringer Mannheim). (d) Primary lymphocytes treated with Tat protein for 72 h displayed activation of cyclin E associated cdk2, a late G1 kinase. Lanes: 1, control cells; 2, cells treated with Tat (24 pmol ml−1); 3, cells treated with PHA (20 μg ml−1). In a–c, PBMCs were treated with Tat protein (24 pmol ml−1), or PHA (20 μg ml−1) plus 20 units ml−1 recombinant human interleukin 2 (rIL-2; Boehringer Mannheim) for 72 h. Similar results were observed in purified T cells. In d, PBMCs were similarly treated as in b and c.

Article Snippet: In a – c , PBMCs were treated with Tat protein (24 pmol ml −1 ), or PHA (20 μg ml −1 ) plus 20 units ml −1 recombinant human interleukin 2 (rIL-2; Boehringer Mannheim) for 72 h. Similar results were observed in purified T cells.

Techniques: Activation Assay, Recombinant, Purification