Journal: The Journal of Biological Chemistry

Article Title: Membrane-bound Trafficking Regulates Nuclear Transport of Integral Epidermal Growth Factor Receptor (EGFR) and ErbB-2 *

doi: 10.1074/jbc.M111.314799

Figure Lengend Snippet: Cell surface ErbB-2, but not FGFR-1, is localized to the INM. A , cell surface ErbB-2 was localized to the INM. The INM-sucrose fractions were isolated from MDA-MB-453 cells treated with or without biotin using sucrose gradient purification and subjected

Article Snippet: We also performed confocal microscopy to examine the subnuclear localization of ErbB-2 (Ab-3, Calbiochem) in the digitonin pretreatment assay in MDA-MB-453 cells and found that nuclear transport of ErbB-2 was still detected after digitonin permeabilization ( ).

Techniques: Isolation, Multiple Displacement Amplification, Purification

Journal: The Journal of Biological Chemistry

Article Title: Membrane-bound Trafficking Regulates Nuclear Transport of Integral Epidermal Growth Factor Receptor (EGFR) and ErbB-2 *

doi: 10.1074/jbc.M111.314799

Figure Lengend Snippet: Cell surface ErbB-2 associates with the translocon Sec61β in the INM. A , INM portions of MDA-MB-453 cells had undetectable cross-contamination during cellular fractionation. Biotinylated cell surface proteins were subjected to cellular fractionation

Article Snippet: We also performed confocal microscopy to examine the subnuclear localization of ErbB-2 (Ab-3, Calbiochem) in the digitonin pretreatment assay in MDA-MB-453 cells and found that nuclear transport of ErbB-2 was still detected after digitonin permeabilization ( ).

Techniques: Multiple Displacement Amplification, Cell Fractionation

Journal: Molecular and Cellular Biology

Article Title: Redistribution of Glycolipid Raft Domain Components Induces Insulin-Mimetic Signaling in Rat Adipocytes

doi: 10.1128/MCB.21.14.4553-4567.2001

Figure Lengend Snippet: CSDP impairs PIG-induced tyrosine phosphorylation of pp59 Lyn (A) and tyrosine phosphorylation of pp125 Fak and IRS-1 and glucose transport (B). Isolated rat adipocytes were electroporated in the absence of CSDP or presence of increasing concentrations of CSDP and then incubated (20 min, 37°C) with increasing concentrations of PIG 41, 37, and 7 (A) or 2 μM PIG 41 (B). From portions of the cells, pp59 Lyn (A, B) and pp125 Fak and IRS-1 (B) were immunoprecipitated (IP) from total-cell lysates and were immunoblotted (IB) for phosphotyrosine (pY). (A) Shown are phosphorimages of a typical experiment that was repeated three times, with similar results. (B) Other portions of the cells were assayed for 2-deoxyglucose transport. Shown are quantitative evaluations of four different adipocyte incubations with measurements in duplicate, and results are given as the percent of PIG response (mean ± standard deviation), set at 100% in the absence of CSDP.

Article Snippet: For immunoblotting, antibodies against phosphotyrosine (clone PY20) were from Transduction Laboratories; those against phosphotyrosine (clone 4G10) were from Upstate Biotechnology; those against synthetic peptide corresponding to the carboxy-terminal sequence comprising amino acids 1223 to 1235 of rat IRS-1 (rabbit) were from Suzanne Dalle (Humbold University, Berlin, Germany); antibodies against paxillin (clone 165), caveolin (rabbit), pp59Lyn (clone 32), and pp125Fak (clone 197) were from Transduction Laboratories; those against human integrin β1 and IRβ were from Upstate Biotechnology; and those against rat glucose transporter isoform 1 (Glut1) and Glut4 (rabbit) were from Biotrend.

Techniques: Isolation, Incubation, Immunoprecipitation, Standard Deviation

Journal: Molecular and Cellular Biology

Article Title: Redistribution of Glycolipid Raft Domain Components Induces Insulin-Mimetic Signaling in Rat Adipocytes

doi: 10.1128/MCB.21.14.4553-4567.2001

Figure Lengend Snippet: Intracellular CBDP causes dissociation of pp59 Lyn and caveolin in DIG. Isolated rat adipocytes were electroporated in the absence or presence of different concentrations of wild-type or mutant CBDP derived from pp59 Lyn . From portions of total-cell lysates, DIG were prepared (detergent method) and used for immunoprecipitation (IP) (dissociating conditions) of pp59 Lyn and caveolin. From other portions of the lysates, pp125 Fak and IRS-1 were immunoprecipitated. The immunoprecipitates were immunoblotted (IB) for caveolin, pp59 Lyn , and phosphotyrosine by immunoblotting using chemiluminescent detection. Shown are phosphorimages of a typical experiment that was repeated three times, with similar results.

Article Snippet: For immunoblotting, antibodies against phosphotyrosine (clone PY20) were from Transduction Laboratories; those against phosphotyrosine (clone 4G10) were from Upstate Biotechnology; those against synthetic peptide corresponding to the carboxy-terminal sequence comprising amino acids 1223 to 1235 of rat IRS-1 (rabbit) were from Suzanne Dalle (Humbold University, Berlin, Germany); antibodies against paxillin (clone 165), caveolin (rabbit), pp59Lyn (clone 32), and pp125Fak (clone 197) were from Transduction Laboratories; those against human integrin β1 and IRβ were from Upstate Biotechnology; and those against rat glucose transporter isoform 1 (Glut1) and Glut4 (rabbit) were from Biotrend.

Techniques: Isolation, Mutagenesis, Derivative Assay, Immunoprecipitation

Journal: Molecular and Cellular Biology

Article Title: Redistribution of Glycolipid Raft Domain Components Induces Insulin-Mimetic Signaling in Rat Adipocytes

doi: 10.1128/MCB.21.14.4553-4567.2001

Figure Lengend Snippet: CBDP and PIG affect pp59 Lyn tyrosine phosphorylation and its activity and interaction with caveolin in a similar fashion. Isolated rat adipocytes were electroporated or incubated (20 min, 37°C) in the absence or presence of increasing concentrations of wild-type or mutant CBDP derived from pp59 Lyn (A, B) or PIG 41 and PIG 1 (B). From portions of total-cell lysates, DIG were prepared (detergent method) and used for direct immunoblotting (IB) of pp59 Lyn (A) or immunoprecipitation (IP) (nondissociating conditions) of caveolin (B). From other portions of the lysates, caveolin (A) and pp59 Lyn (B) were immunoprecipitated. The immunoprecipitates were immunoblotted for pp59 Lyn and phosphotyrosine (pY) or measured for pp59 Lyn activity by the immune complex kinase assay. (A) Shown are phosphorimages of a typical experiment that was repeated two times, with similar results. (B) Quantitative evaluations of three different adipocyte incubations with measurements in triplicate each are given as the percentage of maximum or fold stimulation (mean plus standard deviation), with basal values set at 100% or 1.

Article Snippet: For immunoblotting, antibodies against phosphotyrosine (clone PY20) were from Transduction Laboratories; those against phosphotyrosine (clone 4G10) were from Upstate Biotechnology; those against synthetic peptide corresponding to the carboxy-terminal sequence comprising amino acids 1223 to 1235 of rat IRS-1 (rabbit) were from Suzanne Dalle (Humbold University, Berlin, Germany); antibodies against paxillin (clone 165), caveolin (rabbit), pp59Lyn (clone 32), and pp125Fak (clone 197) were from Transduction Laboratories; those against human integrin β1 and IRβ were from Upstate Biotechnology; and those against rat glucose transporter isoform 1 (Glut1) and Glut4 (rabbit) were from Biotrend.

Techniques: Activity Assay, Isolation, Incubation, Mutagenesis, Derivative Assay, Immunoprecipitation, Immune Complex Kinase Assay, Standard Deviation

Journal: Molecular and Cellular Biology

Article Title: Redistribution of Glycolipid Raft Domain Components Induces Insulin-Mimetic Signaling in Rat Adipocytes

doi: 10.1128/MCB.21.14.4553-4567.2001

Figure Lengend Snippet: Intracellular CBDP causes stimulation of IRS-1 tyrosine phosphorylation, PI-3′K, and glucose transport. Isolated rat adipocytes were electroporated in the absence or presence of increasing concentrations of wild-type CBDP derived from pp59 Lyn . From portions of the cells, total lysates were prepared and used for immunoprecipitation (IP) of IRS-1. The immunoprecipitates were immunoblotted (IB) for phosphotyrosine (pY) or assessed for associated PI-3′K activity by the immune complex kinase assay. Other portions of the cells were assayed for 2-deoxyglucose transport. Quantitative evaluations of three different adipocyte incubations with measurements in quadruplicate each are given as fold stimulation (mean ± standard deviation), with basal values set at 1.

Article Snippet: For immunoblotting, antibodies against phosphotyrosine (clone PY20) were from Transduction Laboratories; those against phosphotyrosine (clone 4G10) were from Upstate Biotechnology; those against synthetic peptide corresponding to the carboxy-terminal sequence comprising amino acids 1223 to 1235 of rat IRS-1 (rabbit) were from Suzanne Dalle (Humbold University, Berlin, Germany); antibodies against paxillin (clone 165), caveolin (rabbit), pp59Lyn (clone 32), and pp125Fak (clone 197) were from Transduction Laboratories; those against human integrin β1 and IRβ were from Upstate Biotechnology; and those against rat glucose transporter isoform 1 (Glut1) and Glut4 (rabbit) were from Biotrend.

Techniques: Isolation, Derivative Assay, Immunoprecipitation, Activity Assay, Immune Complex Kinase Assay, Standard Deviation

Journal: Molecular and Cellular Biology

Article Title: Redistribution of Glycolipid Raft Domain Components Induces Insulin-Mimetic Signaling in Rat Adipocytes

doi: 10.1128/MCB.21.14.4553-4567.2001

Figure Lengend Snippet: PIG-induced dissociation of Gce1, pp125 Fak , and pp59 Lyn from caveolin correlates to caveolin tyrosine phosphorylation. Isolated rat adipocytes were incubated (20 min, 37°C) with increasing concentrations of PIG 41, 37, or 1. From total-cell lysates, caveolin was immunoprecipitated (IP). The immunoprecipitates were assayed for the presence of Gce1 by photoaffinity labeling or for the presence of pp125 Fak , pp59 Lyn , phosphotyrosine (pY), and caveolin by immunoblotting (IB). Shown are quantitative evaluations of three different adipocyte incubations with measurements taken in quadruplicate, and results are given as a ratio of the relative amount of caveolin (mean ± standard deviation) or as the fold stimulation (mean ± standard deviation), with basal values set at 1.

Article Snippet: For immunoblotting, antibodies against phosphotyrosine (clone PY20) were from Transduction Laboratories; those against phosphotyrosine (clone 4G10) were from Upstate Biotechnology; those against synthetic peptide corresponding to the carboxy-terminal sequence comprising amino acids 1223 to 1235 of rat IRS-1 (rabbit) were from Suzanne Dalle (Humbold University, Berlin, Germany); antibodies against paxillin (clone 165), caveolin (rabbit), pp59Lyn (clone 32), and pp125Fak (clone 197) were from Transduction Laboratories; those against human integrin β1 and IRβ were from Upstate Biotechnology; and those against rat glucose transporter isoform 1 (Glut1) and Glut4 (rabbit) were from Biotrend.

Techniques: Isolation, Incubation, Immunoprecipitation, Labeling, Standard Deviation

Journal: The Journal of Experimental Medicine

Article Title: Cbl-b Positively Regulates Btk-mediated Activation of Phospholipase C-?2 in B Cells

doi: 10.1084/jem.20020068

Figure Lengend Snippet: Characterization of the PLC-γ2/Ca 2+ signaling, Rap1 activation, and MAPKs activation in Cbl-b–deficient DT40 cells. (A) Ca 2+ mobilization in wild-type DT40 cells (red) and Cbl-b–deficient cells, and clones C3-1 (blue) and C3-3 (green). [Ca 2+ ] i were monitored by spectrophotometer after stimulation with 2 μg/ml M4 or 0.5 μg/ml ionomycin. Ca 2+ release from intracellular Ca 2+ store was monitored after stimulation with 2 μg/ml M4 in the presence of 1 mM EGTA (+EGTA). Ca 2+ was restored to the media to evaluate extracellular Ca 2+ influx. Arrows indicate the time point of the addition of M4 (α − μ), ionomycin, and CaCl 2 . Similar results were obtained using two other Cbl-b–deficient clones. (B) IP 3 production in wild-type and Cbl-b–deficient DT40 cells. 2 × 10 6 cells were stimulated with 2 μg M4 for the indicated time period and IP 3 production was measured. Results are the mean ± SEM of three independent experiments. (C) Tyrosine phosphorylation of PLC-γ2 in wild-type and Cbl-b–deficient DT40 cells. 10 7 cells were lysed in 1% NP-40 lysis buffer at the indicated time points after stimulation of 4 μg/ml M4. Anti–PLC-γ2 immunoprecipitates from the lysates were subjected to Western blot analysis with anti-phosphotyrosine mAb (top) and anti–PLC-γ2 Ab (bottom). (D) Rap1 activation in wild-type and Cbl-b–deficient DT40 cells. 1.25 × 10 7 cells were lysed in 1% NP-40 lysis buffer at the indicated time points after stimulation of 5 μg/ml M4. Cell lysates were precipitated with GST-RalGDS(RBD) immobilized on glutathione Sepharose, and bound proteins were analyzed by anti-Rap1 immunoblotting. (E–G) BCR-induced ERK2, p38, and JNK activation in wild-type and Cbl-b–deficient DT40 cells. 2.5 × 10 7 cells were lysed at the indicated time points after stimulation of 4 μg/ml M4. (E) Anti-ERK2, (F) anti-p38, or (G) anti-JNK1 immunoprecipitates from the lysates were subjected to kinase reaction using GST-Elk, GST-ATF2, or GST–c-Jun as an exogenous substrate, respectively. The protein levels in the immunoprecipitates are shown in E–G. All experiments were performed more than three times.

Article Snippet: The following Abs were purchased: anti–Cbl-b (C-20), anti–Cbl-b (G-1), anti–mouse PLC-γ2, anti-Rap1, anti–extracellular signal-regulated kinase (ERK)2, and anti-p38 (Santa Cruz Biotechnology, Inc.); anti-JNK1 (BD PharMingen), anti-T7 epitope (Novagen), anti-phosphotyrosine (4G10; Upstate Biotechnology), anti–α-tubulin (Oncogene Research Products); and anti–mouse IgM (μ chain specific; Sigma-Aldrich).

Techniques: Planar Chromatography, Activation Assay, Clone Assay, Spectrophotometry, Lysis, Western Blot

Journal: The Journal of Experimental Medicine

Article Title: Cbl-b Positively Regulates Btk-mediated Activation of Phospholipase C-?2 in B Cells

doi: 10.1084/jem.20020068

Figure Lengend Snippet: Role of Cbl-b on the PLC-γ2/Ca 2+ pathway in mouse B cells. (A) Whole cell lysate (5 × 10 5 cells) from parental and Cbl-b–overexpressing (C85-11 and C85-12) WEHI-231 mouse B cells were analyzed by Western blot analysis using anti–Cbl-b (G-1) mAb. (B) Parental and Cbl-b–overexpressing (C85-11) WEHI-231 cells (3 × 10 6 ) were stimulated with anti-IgM (10 μg/10 7 cells) for 3 min. Anti–PLC-γ2 immunoprecipitates from the lysates were subjected to Western blot analysis with anti-phosphotyrosine mAb (top) and anti–PLC-γ2 mAb (bottom). (C) BCR-induced Ca 2+ mobilization in parental and Cbl-b–overexpressing (C85-11 and C85-12) WEHI-231 cells. [Ca 2+ ] i were monitored by spectrophotometer after stimulation with 2 μg/ml anti-IgM. Arrows indicate the time point of the addition of anti-IgM (α − μ). (D) BCR-induced Ca 2+ mobilization in B cells from wild-type (Cbl-b +/+ ) and Cbl-b–deficient (Cbl-b −/− ) mice. Splenocytes were loaded with Indo-1, labeled with B220-FITC, and stimulated with anti-IgM (10 μg/10 7 cells). Data are presented as the median ratio of Ca 2+ bound to Ca 2+ -free Indo-1 fluorescence of B220 + cells as measured by flow cytometry. (E) 3 × 10 6 splenic B cells of wild-type (Cbl-b +/+ ) or Cbl-b–deficient (Cbl-b −/− ) mice were stimulated with anti-IgM for 1 min. Anti–PLC-γ2 immunoprecipitates from the lysates were subjected to Western blot analysis with anti-phosphotyrosine mAb (top) and anti–PLC-γ2 mAb (bottom). All experiments were performed more than three times.

Article Snippet: The following Abs were purchased: anti–Cbl-b (C-20), anti–Cbl-b (G-1), anti–mouse PLC-γ2, anti-Rap1, anti–extracellular signal-regulated kinase (ERK)2, and anti-p38 (Santa Cruz Biotechnology, Inc.); anti-JNK1 (BD PharMingen), anti-T7 epitope (Novagen), anti-phosphotyrosine (4G10; Upstate Biotechnology), anti–α-tubulin (Oncogene Research Products); and anti–mouse IgM (μ chain specific; Sigma-Aldrich).

Techniques: Planar Chromatography, Western Blot, Spectrophotometry, Mouse Assay, Labeling, Fluorescence, Flow Cytometry, Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Cbl-b Positively Regulates Btk-mediated Activation of Phospholipase C-?2 in B Cells

doi: 10.1084/jem.20020068

Figure Lengend Snippet: BCR-induced activation of Syk and Btk. (A) Tyrosine phosphorylation profiles of wild-type and Cbl-b–deficient (C3-3) DT40 cells. Whole cell lysates were prepared at the indicated time points after stimulation of 4 μg/ml M4. The lysates were subjected to Western blot analysis with anti-phosphotyrosine mAb. (B) Tyrosine phosphorylation and kinase activity of Syk. Wild-type or Cbl-b–deficient (C3-3) DT40 cells were lysed at the indicated time points after stimulation of 4 μg/ml M4. Anti-Syk immunoprecipitates from the lysates were equally divided, and aliquots (one third of the lysates) were subjected to in vitro kinase assay with GST-Igα as a substrate (top), and the remaining immunoprecipitates were analyzed by Western blot analysis with anti-phosphotyrosine mAb (middle) and anti-Syk Ab (bottom). (C) Kinase activity of Btk. Wild-type or Cbl-b–deficient DT40 cells expressing T7-tagged Btk were lysed at the indicated time points after stimulation of 4 μg/ml M4. Anti-T7 immunoprecipitates from the lysates were equally divided, and half of them were subjected to in vitro kinase assay with enolase as a substrate (top), and the remaining half were analyzed by Western blot analysis with anti-T7 mAb (bottom). (D) Tyrosine phosphorylation of Btk. Wild-type or Cbl-b–deficient DT40 cells expressing T7-tagged Btk were lysed in 0.2% NP-40 lysis buffer at the indicated time points after stimulation of 4 μg/ml M4. Anti-T7 immunoprecipitates from the lysates were used for Western blot analysis with anti-phosphotyrosine mAb (top) and anti-T7 mAb (bottom). (E) Tyrosine phosphorylation of BLNK. Wild-type or Cbl-b–deficient (C3-3) DT40 cells (5 × 10 6 ) were lysed in 1% NP-40 lysis buffer at the indicated time points after stimulation of 4 μg/ml M4. Anti-BLNK immunoprecipitates from the lysates were subjected to Western blot analysis with anti-phosphotyrosine mAb (top) and anti-BLNK Ab (bottom). All experiments were performed more than three times.

Article Snippet: The following Abs were purchased: anti–Cbl-b (C-20), anti–Cbl-b (G-1), anti–mouse PLC-γ2, anti-Rap1, anti–extracellular signal-regulated kinase (ERK)2, and anti-p38 (Santa Cruz Biotechnology, Inc.); anti-JNK1 (BD PharMingen), anti-T7 epitope (Novagen), anti-phosphotyrosine (4G10; Upstate Biotechnology), anti–α-tubulin (Oncogene Research Products); and anti–mouse IgM (μ chain specific; Sigma-Aldrich).

Techniques: Activation Assay, Western Blot, Activity Assay, In Vitro, Kinase Assay, Expressing, Lysis

Journal: The Journal of Experimental Medicine

Article Title: Cbl-b Positively Regulates Btk-mediated Activation of Phospholipase C-?2 in B Cells

doi: 10.1084/jem.20020068

Figure Lengend Snippet: BCR-induced association of Cbl-b, Btk, BLNK, and PLC-γ2 and their translocation into GEMs. (A) Interaction of BLNK, PLC-γ2, or Cbl-b with Btk. Wild-type or Cbl-b–deficient DT40 cells expressing T7-tagged Btk were lysed in 0.2% NP-40 lysis buffer at the indicated time points after stimulation of 4 μg/ml M4. Anti-T7 immunoprecipitates from the lysates were subjected to Western blot analysis with anti–PLC-γ2 Ab, anti–Cbl-b (C-20) Ab, anti-BLNK Ab, and anti-T7 mAb. (B) BCR-induced interaction between Cbl-b and PLC-γ2. Wild-type or Cbl-b–deficient (C3-3) DT40 cells were lysed in 1% NP-40 lysis buffer at the indicated time points after stimulation of 4 μg/ml M4. Anti–Cbl-b (C-20) immunoprecipitates from the lysates were subjected to Western blot analysis with anti–PLC-γ2 Ab (top) and anti–Cbl-b (C-20) Ab (bottom). (C) BCR-induced interaction between Cbl-b and BLNK. Wild-type or Cbl-b–deficient (C3-3) DT40 cells were lysed in 1% NP-40 lysis buffer at the indicated time points after stimulation of 4 μg/ml M4. Anti–Cbl-b (C-20) immunoprecipitates from the lysates were subjected to Western blot analysis with anti-BLNK Ab (top) and anti–Cbl-b (C-20) Ab (bottom). (D) Wild-type DT40 cells expressing T7-tagged Btk were stimulated with 8 μg/ml M4 for 3 min. Cell lysates with or without immunodepletion of Btk using anti-T7 mAb were immunoprecipitated with anti–Cbl-b (C-20) Ab (lanes 1 and 2) or anti-T7 mAb (lane 3). Immunoprecipitates or whole cell lysates were subjected to Western blot analysis with anti–PLC-γ2 Ab, anti–Cbl-b (C-20) Ab, and anti-T7 mAb. (E) Wild-type or Cbl-b–deficient DT40 cells expressing T7-tagged Btk, unstimulated (−) or stimulated (+) with 8 μg/ml M4 for 3 min, were lysed and fractionated by the sucrose gradient (top). Fractions (30 μl/lane; numbered from 5 to 40% density) were resolved by SDS-PAGE and subjected to Western blot analysis with anti–α-tubulin mAb or anti-Lyn Ab (bottom left). Anti–Cbl-b immunoprecipitates from fractions 3 and 4 (GEMs), or fraction 11 (Triton soluble) of the sucrose gradient, were subjected to Western blot analysis with anti–Cbl-b Ab (C-20), anti-BLNK Ab, anti–PLC-γ2 Ab, and anti-T7 mAb (bottom right). Whole cell lysates, anti-T7 immunoprecipitates, and anti-BLNK immunoprecipitates from fractions 3, 4, and 11 of the sucrose gradient were subjected to Western blot analysis with anti–PLC-γ2 Ab, anti-T7 mAb, anti-BLNK Ab, and anti–Cbl-b Ab (C-20). (F) Wild-type, Cbl-b– (C3-3), or Cbl-deficient DT40 cells were lysed in 1% NP-40 lysis buffer at the indicated time points after stimulation of 4 μg/ml M4. Anti–PLC-γ2 immunoprecipitates from the lysates were used for Western blot analysis with anti-BLNK Ab (top) and anti–PLC-γ2 Ab (bottom). (G) Tyrosine phosphorylation of Cbl in wild-type and Cbl-b–deficient DT40 cells. 10 7 cells were lysed in 1% NP-40 lysis buffer at the indicated time points after stimulation of 4 μg/ml M4. Anti-Cbl immunoprecipitates from the lysates were subjected to Western blot analysis with anti-phosphotyrosine mAb (top) and anti-Cbl Ab (bottom). All experiments were performed more than three times.

Article Snippet: The following Abs were purchased: anti–Cbl-b (C-20), anti–Cbl-b (G-1), anti–mouse PLC-γ2, anti-Rap1, anti–extracellular signal-regulated kinase (ERK)2, and anti-p38 (Santa Cruz Biotechnology, Inc.); anti-JNK1 (BD PharMingen), anti-T7 epitope (Novagen), anti-phosphotyrosine (4G10; Upstate Biotechnology), anti–α-tubulin (Oncogene Research Products); and anti–mouse IgM (μ chain specific; Sigma-Aldrich).

Techniques: Planar Chromatography, Translocation Assay, Expressing, Lysis, Western Blot, Immunoprecipitation, SDS Page

Journal: The Journal of Experimental Medicine

Article Title: Cbl-b Positively Regulates Btk-mediated Activation of Phospholipase C-?2 in B Cells

doi: 10.1084/jem.20020068

Figure Lengend Snippet: Tyrosine phosphorylation of Cbl-b and generation of Cbl-b–deficient DT40 B cells. (A) At the indicated time points after stimulation of 8 μg/ml M4, wild-type, Lyn-, Syk-, or Btk-deficient DT40 cells (2.5 × 10 7 ) were lysed in 1% NP-40 lysis buffer containing 10 mg/ml digitonin. Anti–Cbl-b (C-20) immunoprecipitates from the lysates were subjected to Western blot analysis with anti-phosphotyrosine mAb (top) and anti–Cbl-b (C-20) Ab (bottom). (B) Structure of the chicken cbl-b allele, the targeting vector, and the mutated allele. Restriction sites for EcoRI (E) are indicated. (C) Southern blot analysis of wild-type and targeted DT40 cells. EcoRI-digested genomic DNAs were separated on an agarose gel, blotted, and hybridized with the chicken cbl-b cDNA probe (B, 5′ probe). (D) Northern blot analysis of wild-type and Cbl-b–deficient DT40 cells using chicken cDNA probe for cbl-b (top) or β-actin (bottom). (E) Protein expression of Cbl-b in wild-type and Cbl-b–deficient DT40 cells. Immunoprecipitates with anti–Cbl-b (C-20) Ab were prepared from wild-type and Cbl-b–deficient DT40 cells and subjected to Western blot analysis using anti–Cbl-b (C-20) Ab. (F) BCR expression on the surface of wild-type (wt), Cbl-b–deficient ( cbl-b − , C3-1, and C3-3), and various DT40 derivatives were monitored by flow cytometry. Unstained cells were used as the negative controls (dashed histogram). Wild-type and Cbl-b–deficient cells expressing T7-tagged Btk are indicated as T7-Btk/wt and T7-Btk/ cbl-b − , respectively. Cbl-b–deficient cells (C3-3) expressing wild-type Cbl-b, G298E mutant Cbl-b, COOH-terminal deletion mutant Cbl-b (1-444 amino acids), and C373A mutant Cbl-b are shown as WT/ cbl-b − , GE/ cbl-b − , N/ cbl-b − , and CA/ cbl-b − , respectively. The x and y axes for the histograms indicate fluorescence intensity (four-decade log scales) and relative cell number, respectively.

Article Snippet: The following Abs were purchased: anti–Cbl-b (C-20), anti–Cbl-b (G-1), anti–mouse PLC-γ2, anti-Rap1, anti–extracellular signal-regulated kinase (ERK)2, and anti-p38 (Santa Cruz Biotechnology, Inc.); anti-JNK1 (BD PharMingen), anti-T7 epitope (Novagen), anti-phosphotyrosine (4G10; Upstate Biotechnology), anti–α-tubulin (Oncogene Research Products); and anti–mouse IgM (μ chain specific; Sigma-Aldrich).

Techniques: Lysis, Western Blot, Plasmid Preparation, Southern Blot, Agarose Gel Electrophoresis, Northern Blot, Expressing, Flow Cytometry, Cytometry, Mutagenesis, Fluorescence

Journal: The Journal of Experimental Medicine

Article Title: Cbl-b Positively Regulates Btk-mediated Activation of Phospholipase C-?2 in B Cells

doi: 10.1084/jem.20020068

Figure Lengend Snippet: Effects of mutations in Cbl-b on BCR-induced Ca 2+ mobilization. (A) Schematic representation of the wild-type and mutant Cbl-b proteins. 4H, four-helix bundle; EF, EF hand; SH2, Src homology 2; RF, RING finger; PRO, proline-rich region; LZ, leucine zipper. (B) Expression and tyrosine phosphorylation of wild-type or mutant Cbl-b in Cbl-b–deficient cells. The indicated cells were lysed in 1% NP-40 lysis buffer at 0, 1, and 3 min after stimulation of 4 μg/ml M4. Anti–Cbl-b (G-1) immunoprecipitates from the lysates were subjected to Western blot analysis with anti-phosphotyrosine mAb (top) and anti–Cbl-b (G-1) mAb (bottom). The positions of WT, GE, N, and CA Cbl-b are indicated. (C) BCR-induced Ca 2+ mobilization in wild-type cells, Cbl-b–deficient cells (C3-3), and Cbl-b–deficient cells expressing wild-type Cbl-b (WT/ cbl-b − ), G298E Cbl-b (GE/ cbl-b − ), a COOH-terminal deletion mutant (N/ cbl-b − ), or C373A Cbl-b (CA/ cbl-b − ). [Ca 2+ ] i were monitored by spectrophotometer after stimulation with 2 μg/ml M4 (−EGTA). Ca 2+ release from intracellular Ca 2+ store was monitored after stimulation of 2 μg/ml M4 in the presence of 1 mM EGTA (+EGTA), and Ca 2+ was restored to the media to evaluate extracellular Ca 2+ influx. Arrows indicate the time point of the addition of M4 (α − μ) and CaCl 2 . The representative results from four independent clones of each cell type are shown. (D) Association of Cbl-b with PLC-γ2. The indicated cells were lysed in 1% NP-40 lysis buffer at 0, 1, and 3 min after stimulation of 4 μg/ml M4. Anti–PLC-γ2 immunoprecipitates from the lysates were subjected to Western blot analysis with anti–Cbl-b (G-1) mAb (top) and anti–PLC-γ2 Ab (bottom). The positions of WT, GE, N, and CA Cbl-b are indicated. (E) In vitro binding of Cbl-b with the SH3 domains of Btk and PLC-γ2. WEHI-231 cells overexpressing Cbl-b (C85-11) and unstimulated (−) or stimulated (+) with anti-IgM (10 μg/10 7 cells) for 3 min, were lysed in 1% NP-40 lysis buffer, and the lysates were incubated with the indicated GST fusion proteins immobilized on glutathione Sepharose. The bound proteins or lysates (1/10 input) were separated by SDS-PAGE and immunoblotted with anti–Cbl-b (G-1) mAb (left). Expression of the GST fusion proteins was confirmed by Coomassie staining (right). (C–E) Experiments were performed more than three times.

Article Snippet: The following Abs were purchased: anti–Cbl-b (C-20), anti–Cbl-b (G-1), anti–mouse PLC-γ2, anti-Rap1, anti–extracellular signal-regulated kinase (ERK)2, and anti-p38 (Santa Cruz Biotechnology, Inc.); anti-JNK1 (BD PharMingen), anti-T7 epitope (Novagen), anti-phosphotyrosine (4G10; Upstate Biotechnology), anti–α-tubulin (Oncogene Research Products); and anti–mouse IgM (μ chain specific; Sigma-Aldrich).

Techniques: Mutagenesis, Expressing, Lysis, Western Blot, Spectrophotometry, Clone Assay, Planar Chromatography, In Vitro, Binding Assay, Incubation, SDS Page, Staining

Journal: Molecular Biology of the Cell

Article Title: Cbl-transforming Variants Trigger a Cascade of Molecular Alterations That Lead to Epithelial Mesenchymal Conversion

doi:

Figure Lengend Snippet: Cbl-N associates with phosphoprotein(s) of ∼150 kDa. (A) Lysates prepared from MDCK cells or an MDCK cell line expressing 70z-Cbl (clone 20) were incubated with the GST fusion protein of the amino-terminal portion of c-Cbl (GST-Cbl-N). Associated proteins were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-phosphotyrosine antibody. (B) MDCK cells or MDCK cells overexpressing either the Met receptor (Met) or the chimeric CSF-MET receptor (CSF-MET) were serum starved for 48 h before stimulation with either 100 U/ml HGF (lanes 2, 6, 8, and 12) or 100 ng/ml CSF (lanes 4 and 10). Cell lysates were subject to a pulldown assay with the use of GST-Cbl-N (lanes 1–6) and GST-G306E Cbl-N (lanes 7–12). Proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-phosphotyrosine (upper panel) or anti-Met (lower panel) antibodies.

Article Snippet: Anti-phosphotyrosine antibody (4G10) was obtained from Upstate Biotechnology (Lake Placid, NY).

Techniques: Expressing, Incubation, SDS Page

Journal: Molecular Biology of the Cell

Article Title: Cbl-transforming Variants Trigger a Cascade of Molecular Alterations That Lead to Epithelial Mesenchymal Conversion

doi:

Figure Lengend Snippet: Constitutive phosphorylation of 70z-Cbl when expressed in MDCK cells. (A) Scheme of 70z-Cbl and its mutant variants. PTB/SH2, phosphotyrosine-binding SH2 domain; RF, RING finger domain; Pro, proline-rich domain. (B) 70z-Cbl and its variants were immunoprecipitated from MDCK cell populations, separated on an 8% SDS-PAGE gel, and immunoblotted with anti-HA (upper panel) or anti-phosphotyrosine (lower panel) antibodies. (C) Lysates from MDCK cell populations were incubated with the amino-terminal SH2 domain of p85 (GST-N-SH2 p85; upper panel) or the amino- and carboxyl-terminal SH3 domains of Grb2 (GST-N+C-SH3 Grb2; lower panel). Proteins were separated on an 8% SDS-PAGE gel, transferred to nitrocellulose, and immunoblotted with anti-HA antibody. (D) MDCK cell populations were immunoprecipitated with the indicated antibodies, resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-HA antibody.

Article Snippet: Anti-phosphotyrosine antibody (4G10) was obtained from Upstate Biotechnology (Lake Placid, NY).

Techniques: Mutagenesis, Binding Assay, Immunoprecipitation, SDS Page, Incubation

Journal: Molecular Biology of the Cell

Article Title: Cbl-transforming Variants Trigger a Cascade of Molecular Alterations That Lead to Epithelial Mesenchymal Conversion

doi:

Figure Lengend Snippet: Phosphorylation kinetics of EGF and Met receptors in cells expressing c-Cbl or 70z-Cbl. (A) Whole cell lysates (WCL) of MDCK cell populations expressing vector or 70z-Cbl were resolved on an SDS-PAGE gel and immunoblotted with anti-phosphotyrosine antibody (lanes 2 and 3). As a control for the size of 70z-Cbl, anti-HA immunoprecipitation was performed on cell lysates expressing 70z-Cbl protein (lane 1). Lysates prepared from MDCK cell populations expressing vector or 70z-Cbl were immunoprecipitated with anti-EGF receptor antibody (lanes 4 and 5) or the anti-Met receptor antibodies (lanes 6 and 7), resolved on SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-phosphotyrosine antibody. (B) MDCK cell populations were serum starved for 48 h before stimulation with either 100 U/ml HGF (left and middle panels) or 1 ng/ml EGF (right panel) for the indicated times. Cell lysates were immunoprecipitated with either anti-Met (left and middle panels) or anti-EGFR (right panel) antibody. Proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-phosphotyrosine (left and right panels) and anti-Met (middle panel) antibodies. (C) Cell lines were stimulated with HGF for 15 min, after which cells were washed in serum-free medium and harvested at the indicated times. Whole cell lysates were subjected to immunoblotting with phosphospecific JNK or MAPK antibodies.

Article Snippet: Anti-phosphotyrosine antibody (4G10) was obtained from Upstate Biotechnology (Lake Placid, NY).

Techniques: Expressing, Plasmid Preparation, SDS Page, Immunoprecipitation

Journal: Molecular Biology of the Cell

Article Title: Cbl-transforming Variants Trigger a Cascade of Molecular Alterations That Lead to Epithelial Mesenchymal Conversion

doi:

Figure Lengend Snippet: Overexpression of 70z-Cbl in MDCK cells results in alterations in cell morphology. (A) MDCK cells were serum starved for 48 h and stimulated for the indicated times with 100 U/ml HGF. Equal amounts of cell lysate were immunoprecipitated with either anti-Cbl antibody (upper and lower panels) or anti-phosphotyrosine antibody (middle panel). The proteins were resolved by SDS-PAGE and immunoblotted with anti-phosphotyrosine (upper panel) or anti-Cbl (middle and bottom panels) antibodies. (B) Proteins from whole cell lysates from stable MDCK cell lines were separated by SDS-PAGE and immunoblotted with either anti-Cbl (upper panel) or anti-HA antibody (middle panel). HA-tagged Cbl proteins were immunoprecipitated with anti-HA antibody, separated by SDS-PAGE, and immunoblotted with anti-phosphotyrosine antibody (lower panel). (C) MDCK cell lines expressing vector, c-Cbl (clone 8), or 70z-Cbl (clone 20) were incubated for 24 h with or without 5 U/ml HGF, as indicated, and cell colonies were visualized by light microscopy.

Article Snippet: Anti-phosphotyrosine antibody (4G10) was obtained from Upstate Biotechnology (Lake Placid, NY).

Techniques: Over Expression, Immunoprecipitation, SDS Page, Expressing, Plasmid Preparation, Incubation, Light Microscopy

Journal: BMC rheumatology

Article Title: In vitro and in vivo characterization of the JAK1 selectivity of upadacitinib (ABT-494)

doi: 10.1186/s41927-018-0031-x

Figure Lengend Snippet: A. Upadacitinib modeled in the crystal structure of JAK1. A model of JAK1 complexed to upadacitinib is shown in blue. JAK2 (Protein Data Ban code: 2B7 a ) is overlaid in green (18). b . Chemical structure of (3S,4R)-3-ethyl-4-(3H-imidazo [1, 2-a] pyrrolo [2, 3-e] pyrazin-8-yl)-N-(2,2,2-trifluoroethyl) pyrrolidine-1-carboxamide (upadacitinib)

Article Snippet: Recombinant kinase domain of JAK2 was purchased from Millipore (Burlington, MA).

Techniques:

Journal: Molecular and Cellular Biology

Article Title: p50Cdc37 Can Buffer the Temperature-Sensitive Properties of a Mutant of Hck

doi:

Figure Lengend Snippet: p50 Cdc37ΔCT partially enhances the catalytic activity of ts Hck499F. (A) 293T cells expressing either Flag-p50 Cdc37 or Flag-p50 Cdc37ΔCT were incubated at 33°C for 48 h and then lysed with p50 Cdc37 lysis buffer. Anti-Flag immunoprecipitates (IP) derived from whole-cell lysates (WCLs) were subjected to Western blotting with anti-Hsp90 and anti-Flag antibodies. The WCLs were subjected to Western blotting with an anti-Hsp90 antibody. (B) 293T cells expressing ts Hck499F alone or ts Hck499F together with either Flag-p50 Cdc37 or Flag-p50 Cdc37ΔCT were incubated at 33°C for 48 h and then lysed with NP-40 lysis buffer. WCLs were subjected to Western blotting with anti-phosphotyrosine (α-pY), anti-Hck, and anti-p50 Cdc37 antibodies. The positions of Flag-p50 Cdc37 , Flag-p50 Cdc37ΔCT , and endogenous p50 Cdc37 are shown on the right. (C) 293T cells transiently expressing paxillin either alone or together with ts Hck499F and Flag-p50 Cdc37 , or ts Hck499F and Flag-p50 Cdc37ΔCT , were incubated at 39°C for 48 h and then lysed with NP-40 lysis buffer. Paxillin was immunoprecipitated from aliquots of the WCLs and subjected to Western blotting with anti-pY and anti-paxillin monoclonal antibodies. The WCLs were subjected to Western blotting with anti-p50 Cdc37 antibodies.

Article Snippet: The anti-phosphotyrosine monoclonal antibody (4G10) was from Upstate Biotechnology, Inc.

Techniques: Activity Assay, Expressing, Incubation, Lysis, Derivative Assay, Western Blot, Immunoprecipitation

Journal: Molecular and Cellular Biology

Article Title: p50Cdc37 Can Buffer the Temperature-Sensitive Properties of a Mutant of Hck

doi:

Figure Lengend Snippet: Hsp90 function is required for ts Hck499F activity. (A) 293T cells expressing ts Hck499F and/or Flag-p50 Cdc37 were treated for 4 h at 33°C with 2.5 μM geldanamycin and then lysed with NP-40 lysis buffer. The whole-cell lysates (WCLs) were subjected to Western blotting with anti-phosphotyrosine (α-pY), anti-Hck, anti-Flag, and anti-Hsp90 antibodies. (B) 293T cells expressing ts Hck499F alone or together with either Hsp90 or Flag-p50 Cdc37 , or both Hsp90 and Flag-p50 Cdc37 , were incubated at 33°C for 48 h and then lysed with NP-40 lysis buffer. The WCLs were then subjected to Western blotting with anti-pY, anti-Hck, anti-Hsp90, and anti-Flag antibodies.

Article Snippet: The anti-phosphotyrosine monoclonal antibody (4G10) was from Upstate Biotechnology, Inc.

Techniques: Activity Assay, Expressing, Lysis, Western Blot, Incubation

Journal: Molecular and Cellular Biology

Article Title: p50Cdc37 Can Buffer the Temperature-Sensitive Properties of a Mutant of Hck

doi:

Figure Lengend Snippet: Conversion of Hck into a temperature-sensitive kinase. (A) 293T cells transiently expressing Hck499F or ts Hck499F were incubated at the indicated temperature for 48 h and then lysed with NP-40 lysis buffer. Aliquots of the whole-cell lysates (WCLs) were sequentially Western blotted with anti-phosphotyrosine (α-pY) and Hck (α-Hck) monoclonal antibodies. The positions of molecular weight markers (in thousands) are shown on the right. The relative specific activities (Hck Sp. Act.) of Hck499F and ts Hck499F are shown at the bottom. The specific activity of ts Hck499F was given an arbitrary value of 1.0. (B) Hck was immunoprecipitated (IP) from aliquots of the WCLs and subjected to an in vitro autophosphorylation reaction. (C) 293T cells transiently expressing paxillin alone, or together with either Hck499F or ts Hck499F, were incubated at the indicated temperature for 48 h and then lysed with NP-40 lysis buffer. Paxillin was immunoprecipitated from aliquots of the WCLs and subjected to Western blotting with anti-pY and anti-paxillin monoclonal antibodies.

Article Snippet: The anti-phosphotyrosine monoclonal antibody (4G10) was from Upstate Biotechnology, Inc.

Techniques: Expressing, Incubation, Lysis, Western Blot, Molecular Weight, Activated Clotting Time Assay, Activity Assay, Immunoprecipitation, In Vitro

Journal: Molecular and Cellular Biology

Article Title: p50Cdc37 Can Buffer the Temperature-Sensitive Properties of a Mutant of Hck

doi:

Figure Lengend Snippet: p50 Cdc37 rescues the catalytic activity of ts Hck499F at 33°C. (A) 293T cells transiently expressing Hck499F or ts Hck499F alone or together with Flag-p50 Cdc37 were incubated at 33°C for 48 h and then lysed with NP-40 lysis buffer. Aliquots of the whole-cell lysates (WCLs) were then sequentially Western blotted with anti-phosphotyrosine (α-pY), anti-Hck, anti-Flag, and anti-p50 Cdc37 antibodies. (B) 293T cells transiently expressing ts Hck499F and increasing (fivefold) amounts of Flag-p50 Cdc37 were incubated at 33°C for 48 h and then lysed with NP-40 lysis buffer. Aliquots of the WCLs were then sequentially Western blotted with anti-pY, anti-Hck, and anti-Flag monoclonal antibodies. (C) Hck was immunoprecipitated (IP) from aliquots of the WCLs shown in panel B and subjected to an in vitro autophosphorylation reaction. (D) 293T cells transiently coexpressing paxillin and ts Hck499F, together with increasing amounts of Flag-p50 Cdc37 , were incubated at 33°C for 48 h and then lysed with NP-40 lysis buffer. Paxillin was immunoprecipitated from aliquots of the WCLs and subjected to Western blotting with anti-pY and anti-paxillin monoclonal antibodies.

Article Snippet: The anti-phosphotyrosine monoclonal antibody (4G10) was from Upstate Biotechnology, Inc.

Techniques: Activity Assay, Expressing, Incubation, Lysis, Western Blot, Immunoprecipitation, In Vitro

Journal: Molecular and Cellular Biology

Article Title: p50Cdc37 Can Buffer the Temperature-Sensitive Properties of a Mutant of Hck

doi:

Figure Lengend Snippet: p50 Cdc37 partially rescues the catalytic activity of ts Hck499F at 37 and 39°C. (A) 293T cells transiently expressing ts Hck499F alone or together with Flag-p50 Cdc37 were incubated at the indicated temperatures for 48 h and then lysed with NP-40 lysis buffer. Aliquots of the whole-cell lysates (WCLs) were then sequentially Western blotted with anti-phosphotyrosine (α-pY), anti-Hck, and anti-Flag antibodies. (B) 293T cells transiently expressing paxillin either alone or together with ts Hck499F and Flag-p50 Cdc37 were incubated at the indicated temperature for 48 h and then lysed with NP-40 lysis buffer. Paxillin was immunoprecipitated (IP) from aliquots of the WCLs and subjected to Western blotting with anti-pY and anti-paxillin monoclonal antibodies. The WCLs were subjected to Western blotting with an anti-Flag monoclonal antibody.

Article Snippet: The anti-phosphotyrosine monoclonal antibody (4G10) was from Upstate Biotechnology, Inc.

Techniques: Activity Assay, Expressing, Incubation, Lysis, Western Blot, Immunoprecipitation

Journal: Molecular and Cellular Biology

Article Title: p50Cdc37 Can Buffer the Temperature-Sensitive Properties of a Mutant of Hck

doi:

Figure Lengend Snippet: Posttranslational stabilization of ts Hck499F by p50 Cdc37 . 293T cells transiently expressing paxillin alone, or together with ts Hck499F and/or Flag-p50 Cdc37 , were incubated at 39°C for 48 h and then treated with either ethanol vehicle (−CHX) (A) or 100 μg of cycloheximide/ml (+CHX) (B) for 90 min. The cells were then either left at 39°C or shifted to 33°C for 6 h prior to lysing with NP-40 lysis buffer. Paxillin was immunoprecipitated (IP) from aliquots of the whole-cell lysates (WCLs) and subjected to Western blotting with anti-phosphotyrosine (α-pY) and anti-paxillin monoclonal antibodies. The WCLs were subjected to Western blotting with anti-Hck and anti-Flag antibodies.

Article Snippet: The anti-phosphotyrosine monoclonal antibody (4G10) was from Upstate Biotechnology, Inc.

Techniques: Expressing, Incubation, Lysis, Immunoprecipitation, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Src Catalytic but Not Scaffolding Function Is Needed for Integrin-Regulated Tyrosine Phosphorylation, Cell Migration, and Cell Spreading

doi: 10.1128/MCB.22.8.2427-2440.2002

Figure Lengend Snippet: Src SH2 and SH3 domain mutations reduce binding to FAK, paxillin, and Cas. (A and B) NIH3T3 cells were lysed on the dish (Att) or were harvested with trypsin and lysed in suspension (Sus). These lysates were used for in vitro binding experiments with GST-Src fusion proteins. (A) Levels of FAK and paxillin input in RIPA buffer cell lysates (not shown) or immunoprecipitates (left) were equal. Attachment-induced phosphorylation of immunoprecipitated FAK and paxillin was detected with MAb 4G10 (middle). Attachment-induced binding of FAK and paxillin to GST-SrcSH2 was significantly reduced by the T215W mutation (right). (B) Binding of FAK from Triton buffer lysates to GST-SrcSH3 was not affected by FAK phosphotyrosine levels (left). Binding of FAK (middle) and Cas (right) (from attached cells) to GST-SrcSH3 was significantly reduced by the D99N mutation. The input level of Cas shown (right) is 5% of that used for the binding experiment. (C) Approximately equal amounts of each GST fusion protein were prepared as determined by Coomassie staining, although amounts used for binding assays were adjusted slightly to account for differences. (D through G) SYF cells reconstituted with vector or the indicated Src molecules were harvested with trypsin, washed, and lysed with NP-40 buffer in suspension (Sus) or after replating on FN-coated dishes for 30 min (FN). Whole cell lysates were Western blotted with anti-FAK C20 (D) or anti-Src LA074 (E). Alternatively, Src was immunoprecipitated with LA074 followed by Western blotting with anti-FAK C20 to detect associated FAK (F) or anti-Src LA074 (G). WB, Western blotting; IP, immunoprecipitation.

Article Snippet: The anti-phosphotyrosine MAb 4G10 was purchased from Upstate Biotechnology, Inc.

Techniques: Binding Assay, In Vitro, Immunoprecipitation, Mutagenesis, Staining, Plasmid Preparation, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Src Catalytic but Not Scaffolding Function Is Needed for Integrin-Regulated Tyrosine Phosphorylation, Cell Migration, and Cell Spreading

doi: 10.1128/MCB.22.8.2427-2440.2002

Figure Lengend Snippet: Activity of Src SH2 and SH3 domain mutants. SYF cells stably expressing wtSrc, an SH2 domain mutant (T215W) or an SH3 domain mutant (D99N), or an activated mutant (Y527F) were established. (A) Phase-contrast images of near-confluent cell cultures at ×100 magnification (scale bar, ∼200 μm). (B) Lysates using Triton buffer were generated from similar cultures, and total proteins were Western blotted with anti-phosphotyrosine MAb 4G10. The positions of molecular mass markers (in kDa) are shown on the right. An asterisk indicates the position of Src. To detect the activation level of each form of Src, anti-Src immunoprecipitates (using MAb LA074) from growing cells were Western blotted with anti-Src-pY416 (C) or anti-Src LA074 (D).

Article Snippet: The anti-phosphotyrosine MAb 4G10 was purchased from Upstate Biotechnology, Inc.

Techniques: Activity Assay, Stable Transfection, Expressing, Mutagenesis, Generated, Western Blot, Activation Assay

Journal: Molecular and Cellular Biology

Article Title: Src Catalytic but Not Scaffolding Function Is Needed for Integrin-Regulated Tyrosine Phosphorylation, Cell Migration, and Cell Spreading

doi: 10.1128/MCB.22.8.2427-2440.2002

Figure Lengend Snippet: Src kinase activity is needed for integrin-dependent FAK phosphorylation. SYF cells were infected using a retroviral system to stably express wtSrc or KD-Src mutants (K295R or D386A) or were infected with virus containing empty vector. Cells were harvested with trypsin, washed, and lysed with Triton buffer in suspension (0 min) or after plating for the indicated times (10, 20, or 30 min) on FN-coated dishes. FAK tyrosine phosphorylation was examined by immunoprecipitating with anti-FAK C20, followed by Western blotting with either anti-FAK C20 to demonstrate equal amounts of FAK in each immunoprecipitate (A), anti-phosphotyrosine 4G10 (B), or the site-specific phosphoantibodies anti-FAK-pY576 (C) or anti-FAK-pY397 (D). Src expression levels were determined by Western blotting cell lysates with anti-Src LA074 (E).

Article Snippet: The anti-phosphotyrosine MAb 4G10 was purchased from Upstate Biotechnology, Inc.

Techniques: Activity Assay, Infection, Stable Transfection, Plasmid Preparation, Western Blot, Expressing

Journal: Molecular and Cellular Biology

Article Title: Src Catalytic but Not Scaffolding Function Is Needed for Integrin-Regulated Tyrosine Phosphorylation, Cell Migration, and Cell Spreading

doi: 10.1128/MCB.22.8.2427-2440.2002

Figure Lengend Snippet: Src scaffolding functions can occur when KD-Src is overexpressed. (A) SYF cells expressing low or high levels of wtSrc or KD-Src (K295R mutant) were established as described in Materials and Methods. Src expression levels were compared with that of endogenous Src in fibroblasts derived from a src +/+ fyn −/− yes −/− ( src +/+ ) mouse embryo. Src expression levels were determined by Western blotting cell lysates (using Triton buffer) with anti-Src LA074 (left) or SRC2 (right). (B through E) Cells expressing low or high levels of wtSrc or KD-Src were harvested with trypsin, washed, and lysed with Triton buffer in suspension (0 min) or after plating on FN-coated dishes for 30 min. (B and C) FAK was immunoprecipitated with anti-KC, followed by Western blotting with anti-phosphotyrosine 4G10 (B) or anti-KC (C). Cas was immunoprecipitated with anti-Cas C20, followed by Western blotting with 4G10 (D) or anti-Cas C20 (E). (F) The cells described above were harvested with Triton lysis buffer in suspension (S) or after plating on FN for 20 min (FN). FAK immunoprecipitates (using anti-FAK C20) were used for in vitro kinase assays using [γ- 32 P]ATP and poly(Glu, Tyr) as a substrate, and kinase activity was normalized against FAK levels (not shown). (G and H) SYF cells lacking or expressing wtSrc were treated with 0 μM (−) or 50 μM (+) sodium vanadate for 5 h to inhibit tyrosine phosphatases. Cells were lysed (with Triton buffer) attached (A) or were harvested with trypsin, washed, and lysed in suspension (S) or after plating on FN for 40 min (FN). (G) FAK immunoprecipitates (using anti-KC) were Western blotted with 4G10. Equal amounts of FAK were detected in each immunoprecipitate (not shown). (H) Whole cell lysates from wtSrc-expressing cells (lysed attached) were Western blotted with 4G10 to demonstrate vanadate-induced phosphotyrosine of many proteins. The positions of molecular mass markers (in kDa) are shown on the right.

Article Snippet: The anti-phosphotyrosine MAb 4G10 was purchased from Upstate Biotechnology, Inc.

Techniques: Scaffolding, Expressing, Mutagenesis, Derivative Assay, Western Blot, Immunoprecipitation, Lysis, In Vitro, Activity Assay

Journal: Molecular and Cellular Biology

Article Title: Src Catalytic but Not Scaffolding Function Is Needed for Integrin-Regulated Tyrosine Phosphorylation, Cell Migration, and Cell Spreading

doi: 10.1128/MCB.22.8.2427-2440.2002

Figure Lengend Snippet: Integrin-induced phosphorylation, migration and spreading require catalytic but not scaffolding functions of Src. SYF cells reconstituted with vector, wtSrc, or the indicated Src mutants (see Results for descriptions) were harvested with trypsin, washed, and resuspended in serum-free medium. (A) Cells were lysed with RIPA buffer in suspension (S) or after plating on fibronectin-coated dishes for 30 min (FN). Total cell lysates were Western blotted with the anti-phosphotyrosine MAb 4G10. The position of molecular mass markers (in kDa) is shown on the left. An asterisk indicates the position of Src. (B) Cell migration towards 10 μg of fibronectin/ml was measured using a chemotaxis chamber. Results shown were compiled from three individual experiments for each cell line. Error bars represent standard errors of the mean. No cells migrated when DMEM without FN was added to the lower wells (not shown). (C and D) Cells were plated on FN-coated dishes at 37°C, and time-lapse phase-contrast images were taken at the indicated times. This experiment was repeated at least three times with similar results. (C) The percentages of cells that had spread at each time point were scored as indicated. (D) Representative images taken 20 min after cell plating. Scale bar, ∼100 μm.

Article Snippet: The anti-phosphotyrosine MAb 4G10 was purchased from Upstate Biotechnology, Inc.

Techniques: Migration, Scaffolding, Plasmid Preparation, Western Blot, Chemotaxis Assay

Journal: Molecular and Cellular Biology

Article Title: Src Catalytic but Not Scaffolding Function Is Needed for Integrin-Regulated Tyrosine Phosphorylation, Cell Migration, and Cell Spreading

doi: 10.1128/MCB.22.8.2427-2440.2002

Figure Lengend Snippet: Regulated Src kinase activity is not required for FAK phosphorylation upon integrin activation. SYF cells expressing the indicated Src molecules (or reconstituted with empty vector) were harvested with trypsin, washed, and lysed in suspension (0 min) or after plating on FN-coated dishes for 10 or 30 min, as indicated. (A and B) Src was immunoprecipitated from RIPA buffer lysates with anti-Src LA074, followed by Western blotting with anti-Src-pY416 (A) or anti-Src SRC2 (B). (C and D) FAK was immunoprecipitated from Triton buffer lysates with anti-FAK C20 followed by Western blotting with anti-phosphotyrosine 4G10 (C) or anti-FAK C20 (D).

Article Snippet: The anti-phosphotyrosine MAb 4G10 was purchased from Upstate Biotechnology, Inc.

Techniques: Activity Assay, Activation Assay, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Src Catalytic but Not Scaffolding Function Is Needed for Integrin-Regulated Tyrosine Phosphorylation, Cell Migration, and Cell Spreading

doi: 10.1128/MCB.22.8.2427-2440.2002

Figure Lengend Snippet: Src-mediated FAK, Cas, and paxillin phosphorylation does not require stable association. SYF cells were reconstituted with vector, wtSrc, or the indicated Src mutants (T215W, D99N, D99N/T215W, or Y527F). Cells were harvested with trypsin, washed, and lysed in suspension (S) or after replating on FN-coated dishes (FN) for 30 (A through D) or 15 (E through H) min. (A through D) FAK was immunoprecipitated from Triton buffer lysates with anti-FAK C20, followed by Western blotting with anti-phosphotyrosine 4G10 (A) or with anti-FAK C20 (B). Some immunoprecipitates were also Western blotted with the site-specific phosphorylation antibody anti-FAK-pY397 (C) or anti-FAK-pY576 (D). Cas (E and F) or paxillin (G and H) immunoprecipitates from RIPA buffer lysates were Western blotted with 4G10 (E and G), anti-Cas (F), or anti-paxillin (H).

Article Snippet: The anti-phosphotyrosine MAb 4G10 was purchased from Upstate Biotechnology, Inc.

Techniques: Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Cell motility and the cytoskeleton

Article Title: A FAK/Src Chimera With Gain-of-Function Properties Promotes Formation of Large Peripheral Adhesions Associated With Dynamic Actin Assembly

doi: 10.1002/cm.20241

Figure Lengend Snippet: FAK/Src F-chimera structure and signaling activity. A : The FAK/Src F-chimera features a substituted Src kinase domain and mutated autophosphorylation site (F397). The remaining major sites of tyrosine phosphorylation are numbered according to their position in the parental proteins. The FAT, FERM, and proline-rich motifs (PR) are also shown. B-E : Immunoblot analysis of total cell lysates of cells expressing WT-FAK, FAK/Src chimeras, or oncogenic Src-F529. FAK/Src chimera variants included “F-chimera” described above, “Y-chimera” with intact Tyr-397 site, and “dead F-chimera” with inactived Src kinase domain. B: Expression and phosphotyrosine profiles. (Top) WT-FAK and chimera expression detected using antibody 331 that recognizes the FAK C-terminal region. (Bottom) Tyrosine-phosphorylated proteins detected using antibody 4G10. Arrows indicate regions of elevated tyrosine phosphorylation in cells expressing the FAK/Src chimeras. Numbers indicate size markers (kDa). C , top: p130Cas substrate domain tyrosine phosphorylation detected using a mixture of pCAS-165, pCAS-249, and pCAS-410 antibodies. C, bottom: Total p130Cas protein. D , top: Paxillin Tyr-118 phosphorylation detected using a phosphospecific antibody. D, bottom: Total paxillin protein. E : Analysis of FAK/Src chimera activity by detection of the phosphorylated Tyr-418 site in the Src kinase domain. Two different regions of the same blot are shown for detecting the FAK/Src chimera proteins (top), or the oncogenic Src-F529 (bottom).

Article Snippet: Anti-phosphotyrosine antibody 4G10 was from Upstate Biotechnology.

Techniques: Activity Assay, Expressing

Journal: Cell motility and the cytoskeleton

Article Title: A FAK/Src Chimera With Gain-of-Function Properties Promotes Formation of Large Peripheral Adhesions Associated With Dynamic Actin Assembly

doi: 10.1002/cm.20241

Figure Lengend Snippet: Adhesion-dependent regulation of the FAK/Src F-chimera. Cells expressing either WT-FAK or the FAK/Src F-chimera were analyzed under normal adherent growth conditions (Att), after holding in suspension for 30 min post-trypsinization (Sus), or after plating onto fibronectin-coated dishes for 30 (Fn30) or 60 (Fn60) min. (Top panels) The proteins were immunoprecipitated using FAK C-20 antibody and assessed by immunoblotting with either FAK C-20 antibody to show protein recovery or 4G10 antibody to show changes in overall tyrosine phosphorylation. (Bottom panels) Changes in specific phosphotyrosine sites in the F-chimera were revealed by immunoblotting with phosphospecific antibodies against FAK (Tyr-861, Tyr-407, or Tyr-925) or Src kinase domain (Tyr-418).

Article Snippet: Anti-phosphotyrosine antibody 4G10 was from Upstate Biotechnology.

Techniques: Expressing, Immunoprecipitation