Journal: The Journal of Neuroscience

Article Title: A Novel Role for Protein Tyrosine Phosphatase SHP1 in Controlling Glial Activation in the Normal and Injured Nervous System

doi: 10.1523/JNEUROSCI.21-03-00865.2001

Figure Lengend Snippet: Astrocytic and microglial reaction in the intact brain during adulthood, in the intact (Con) and axotomized (Ax) facial nucleus in normal, wild-type littermates (WT) and SHP1 mutant, moth-eaten viable mice (MEV). A, GFAP-immunoreactivity in the brainstem of WT and MEV mice at postnatal days 4 (P4) and 14 (P14). Note that GFAP-IR is increased in MEV mice compared with the same brain regions of WT littermates. B, GFAP-IR in the brainstem of WT and MEV mice 3 d after facial nerve transection. Note that GFAP-IR is increased on the control, unoperated side in MEV mice (Con-MEV) (as well as in the whole brain) compared with the same region of normal littermates (Con-WT). C, The morphology of microglia, detected by αMβ2-integrin immunoreactivity 2 and 3 d after facial nerve axotomy. Note that after facial nerve axotomy, the microglial cells in MEV mice are characterized by rounded, ameboid morphology with delayed and decreased ramification. D, Combination of [3H]-thymidine autoradiography (white points) with either GFAP-immunoreactivity (green) or αMβ2-integrin-IR (red) 3 d after facial nerve axotomy. Note that the proliferating cells in MEV mice are exclusively αMβ2-integrin-positive microglia. Scale bars, 100 μm.

Article Snippet: Mouse brain sections (20 μm) from normal littermates and mutant me v /me v mice were incubated with either rat monoclonal antibodies against α M β 2 -integrin (1:6000, Serotec) or rat monoclonal antibodies against GFAP (Zymed).

Techniques: Mutagenesis, Control, Autoradiography

Journal: The Journal of Neuroscience

Article Title: A Novel Role for Protein Tyrosine Phosphatase SHP1 in Controlling Glial Activation in the Normal and Injured Nervous System

doi: 10.1523/JNEUROSCI.21-03-00865.2001

Figure Lengend Snippet: Quantitative effects of reduced SHP1 activity on astrocytic (A) and microglial reaction (B) in the intact and injured brain as well as on axonal outgrowth (C). A, Quantification of GFAP-IR (pixel number) in the intact brain (random brainstem area, nonaxotomized facial nucleus) and 3 d after facial nerve axotomy in wild-type (WT) littermates and MEV mice. Note that only in the uninjured brain regions there is a statistically significant increase for GFAP-IR in MEV mice compared with WT mice (6 sections per each animal; n = 4 animals). B1, Quantification of αMβ2-integrin-positive microglia in intact brain regions (random brainstem area and nonaxotomized facial nucleus). Note that the number of resting microglia is similar in WT and MEV mice. B2, Reduction in the number of proliferating ([3H]-thymidine-labeled) αMβ2-positive microglia in MEV mice 2 d after direct cortical lesion. B3, Reduction in the number of αMβ2-positive microglia and the proliferation rate ([3H]-thymidine-labeled cells) in MEV mice 2 and 3 d after facial nerve axotomy compared with wild-type littermates. The microglial reaction was always quantified in six sections per animal (n = 4). C, Four days after crush near the foramen stylomastoideum, the facial nerve was cut longitudinally and stained for galanin or CGRP, which accumulate in the terminals of the elongating neurites. The average distance between the most distal-labeled growth cone and the crush side was determined for each axonal marker in five tissue sections per animal (n = 6). Both the galanin- and CGRP-positive axonal populations show the same regeneration distance of ∼6 mm at day 4 in the WT littermates and MEV mice. All statistical analyses were performed using a paired, two-tailed Student's t test. Statistically significant changes are indicated byasterisks (*p < 0.05, ***p < 0.001).

Article Snippet: Mouse brain sections (20 μm) from normal littermates and mutant me v /me v mice were incubated with either rat monoclonal antibodies against α M β 2 -integrin (1:6000, Serotec) or rat monoclonal antibodies against GFAP (Zymed).

Techniques: Activity Assay, Labeling, Staining, Marker, Two Tailed Test

Journal:

Article Title: CD98 activation increases surface expression and clustering of ? 1 integrins in MCF-7 cells through FAK/Src- and cytoskeleton-independent mechanisms

doi: 10.3858/emm.2008.40.3.261

Figure Lengend Snippet: CD98 engagement increases the surface expression of β1 integrin. (A) Freshly cultured MCF-7 cells were dispersed and then incubated with anti-CD98 mAb UM7F8 and secondary Ab (blue line) for 1 h. As negative controls, cells were treated with mouse IgG and secondary Ab (red line). FITC-conjugated anti-CD29 mAb was used to measure the expression level of β1 integrin on the cells treated with mAbs as described above. An FITC-conjugated mouse anti-human lgG was used as the negative control (green line). (B) Cell extracts from MCF-7 cells treated as in (A) were analyzed by Western blotting using anti-β1 integrin and anti-actin mAb.

Article Snippet: R-PE-conjugated mouse anti-β 1 integrin mAb (TDM29) was from Cymbus Biotechnology (Eastleigh, Hampshire, UK).

Techniques: Expressing, Cell Culture, Incubation, Negative Control, Western Blot

Journal:

Article Title: CD98 activation increases surface expression and clustering of ? 1 integrins in MCF-7 cells through FAK/Src- and cytoskeleton-independent mechanisms

doi: 10.3858/emm.2008.40.3.261

Figure Lengend Snippet: CD98 cross-linking enhances β1 integrin clustering. (A) MCF-7 cells were incubated with anti-CD98 mAb UM7F8 with or without secondary antibody. To determine whether cross-linking of β1 integrins induces their clustering, cells were treated with anti-β1 integrin mAb 3S3 in the presence of secondary antibody as well. Next, cells treated as described in "Materials and Methods" were analyzed by confocal microscopy. Actin cytoskeleton organization is visualized by staining with phalloidin-FITC whereas β1 integrin clustering with R-PE-conjugated anti-β1 integrin mAb. Images are from a single experiment representative of more than three so performed. Scale bar, 50 µm. Original magnification, × 400. (B) Relative intensities of β1 integrin clusters in Figure 2A were measured with LSM5120 Meta NLO software. Results are values relative to the β1 integrin signal intensity level of untreated controls, designated as 1.

Article Snippet: R-PE-conjugated mouse anti-β 1 integrin mAb (TDM29) was from Cymbus Biotechnology (Eastleigh, Hampshire, UK).

Techniques: Incubation, Confocal Microscopy, Staining, Software

Journal:

Article Title: CD98 activation increases surface expression and clustering of ? 1 integrins in MCF-7 cells through FAK/Src- and cytoskeleton-independent mechanisms

doi: 10.3858/emm.2008.40.3.261

Figure Lengend Snippet: Inhibition of FAK/Src kinases with PP2 blocks CD98-induced cell adhesion, but not surface expression and clustering of β1 integrins in MCF-7 cells. (A) The effect of PP2 and/or Mn2+ on FAK phosphorylation in MCF-7 cells treated with anti-CD98 mAb was determined by immunoprecipitaion assay. MCF-7 cells were incubated with anti-CD98 mAb and PP2 (0.2 µM) or a DMSO vehicle control in the presence or absence of 0.5 µM Mn2+ for 1 h and anti-FAK rabbit polyclonal antibody (C-20) was used to immunoprecipitate FAK from extracts of MCF-7 cells. Immunoprecipitates were blotted and probed with anti-phosphotyrosine mAb (clone PY99) and anti-FAK mAb. (B) The effect of PP2 treatment on cell adhesion rate was determined as described in Materials and Methods. In addition, whether addition of 0.5 µM Mn2+ interferes with the effect of PP2 on cell adhesion was determined by the same way. Results are expressed as mean ± SE of values relative to the adhesion rate of mouse IgG-treated controls, designated as 100%. Asterisks show a significant difference from control as follows: *P < 0.05. Additional statistical comparisons are indicated by lines. (C) MCF-7 cells were incubated with anti-CD98 mAb with or without PP2 (0.2 µM) and then analyzed by flow cytometry using FITC-conjugated anti-human β1 integrin mAb. Data represent the mean ± SE of values relative to mean values of fluorescence intensity of mouse IgG-treated controls, designated as 100%. Asterisks show a significant difference from control as follows: *P < 0.05, ***P < 0.001 (D) Confocal microscopy was performed as described in Figure 2 legend to investigate the effect of PP2 (0.2 µM) on CD98-induced clustering of β1 integrins. Scale bar, 50 µm. Original magnification, × 400.

Article Snippet: R-PE-conjugated mouse anti-β 1 integrin mAb (TDM29) was from Cymbus Biotechnology (Eastleigh, Hampshire, UK).

Techniques: Inhibition, Expressing, Phospho-proteomics, Incubation, Control, Flow Cytometry, Fluorescence, Confocal Microscopy

Journal:

Article Title: CD98 activation increases surface expression and clustering of ? 1 integrins in MCF-7 cells through FAK/Src- and cytoskeleton-independent mechanisms

doi: 10.3858/emm.2008.40.3.261

Figure Lengend Snippet: The effects of dominant-negative variants of FAK on adhesiveness of MCF-7 cells, surface expression and clustering of β1 integrins. (A) MCF-7 cells were stably transfected with dominant-negative mutant FAK constructs (FRNK, Y397F-FAK) or control vector, pcDNA3. The expression levels of endogenous FAK, FRNK and Y397F-FAK were determined by Western blot analysis using anti-FAK polyclonal antibody. (B) The effect of dominant-negative variants of FAK on CD98-induced cell adhesion rate was determined as described in Materials and Methods. In addition, whether addition of 0.5 µM Mn2+ interferes with the effect of PP2 on cell adhesion was determined by the same way. Results are expressed as in Figure 3 (B). *P < 0.05, **P < 0.01 (C) FAK variants- or mock-transfected MCF-7 cells were treatred with anti-CD98 mAb and secondary antibody and then analyzed for expression of β1 integrin through flow cytometry using FITC-conjugated anti-human β1 integrin. Results are expressed as in Figure 3 (C). Statistical comparisons are indicated by lines. **P < 0.01 (D) Confocal microscopy was performed as described above to investigate the effect of dominant-negative variants of FAK on clustering of β1 integrins. Scale bar, 50 µm. Original magnification, × 400.

Article Snippet: R-PE-conjugated mouse anti-β 1 integrin mAb (TDM29) was from Cymbus Biotechnology (Eastleigh, Hampshire, UK).

Techniques: Dominant Negative Mutation, Expressing, Stable Transfection, Transfection, Construct, Control, Plasmid Preparation, Western Blot, Flow Cytometry, Confocal Microscopy

Journal:

Article Title: CD98 activation increases surface expression and clustering of ? 1 integrins in MCF-7 cells through FAK/Src- and cytoskeleton-independent mechanisms

doi: 10.3858/emm.2008.40.3.261

Figure Lengend Snippet: Cytochalasin D or phalloidin treatment inhibits CD98-induced adhesion of MCF-7 cells to fibronectin, but not surface expression and clustering of β1 integrins in MCF-7 cells. (A) MCF-7 cells were incubated with anti-CD98 mAb in the presence or absence of cytochalasin D (4 µM) or phalloidin (10 µM) for 1 h. The effects of DMSO vehicle control are also shown. The effect of cytochalasin D treatment on cell adhesion was determined as described in Materials and Methods. In addition, whether addition of 0.5 µM Mn2+ interferes with the effect of cytochalasin D or phalloidin on cell adhesion was determined by the same way. Results are expressed as Figure 3B. *P < 0.05, **P < 0.01, ***P < 0.001 (B) MCF-7 cells treated with anti-CD98 mAb in the presence or absence of cytochalasin D or phalloidin were analyzed for expression of β1 integrin using flow cytometry. Results are expressed as in Figure 3 (C). **P < 0.01 (C) Confocal microscopy was performed as described above to investigate the effect cytochalasin D or phalloidin of on clustering of β1 integrins. Scale bar, 50 µm. Original magnification, × 400.

Article Snippet: R-PE-conjugated mouse anti-β 1 integrin mAb (TDM29) was from Cymbus Biotechnology (Eastleigh, Hampshire, UK).

Techniques: Expressing, Incubation, Control, Flow Cytometry, Confocal Microscopy