Journal: Immunity

Article Title: Fever Promotes T Lymphocyte Trafficking via a Thermal Sensory Pathway Involving Heat Shock Protein 90 and α4 Integrins

doi: 10.1016/j.immuni.2018.11.013

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Hamster monoclonal FITC anti-β1 integrin , Santa Cruz , Cat# sc-19656; RRID: AB_627005.

Techniques: Virus, Recombinant, Protease Inhibitor, Reverse Transcription, Cell Isolation, shRNA, Software

Journal: Open Biology

Article Title: Spatial activation of ezrin by epidermal growth factor receptor and focal adhesion kinase co-ordinates epithelial cell migration

doi: 10.1098/rsob.210166

Figure Lengend Snippet: EGFR activity is required for keratinocyte adhesion. ( a ) NHKs stably expressing GFP, siRNA-resistant EGFR-GFP (siR-EGFR-GFP) were transfected with control siRNA (siCtrl) or siRNA targeting EGFR (siEGFR) before plating onto laminin. Cells were left to adhere for 40 min before fixation and immunostaining with vinculin (magenta) and F-actin (cyan). Scale bar 10 µm. Phalloidin was used to quantify the average area per cell. N = 3 * p < 0.05, ** p < 0.01; ( b ) NHKs were pre-treated with DMSO or EGFR inhibitor (AG1478) for an hour before re-plating onto laminin in the presence of AG1478. After 40 min, cells were fixed and immunostained with FAK (magenta), EGFR (green) and F-actin (cyan). Phalloidin was used to quantify the average area per cell. N = 4 * p < 0.05, ** p < 0.01; ( c ) NHKs were plated onto laminin before lysates were collected at 0, 20, 40 and 60 min after matrix adhesion. Western blots were probed for phospho-FAK (Y397), FAK, phospho-EGFR (Y1173) and GAPDH. ( d ) Confluent monolayers of NHKs were pre-treated with AG1478 for an hour before wounding. After an additional hour of inhibitor incubation, immunoprecipitation was carried out using an antibody against FAK. Western blot analysis was performed probed with EGFR and FAK. ( e ) Confluent monolayers of NHKs stably expressing eGFP or EGFR-eGFP were pre-treated with AG1478 (AG), PF228 (PF) or DMSO as a negative control. After 1 h, monolayers were wounded and were incubated for an hour with AG1478/PF228 before cell lysates were collected for immunoprecipitation with GFP-TRAP beads. Western blot analysis was carried out probed with FAK, β1 integrin and GFP.

Article Snippet: Primary antibodies for western blotting were mouse monoclonal anti-GAPDH (Chemicom, Mississauga, ON, Canada), anti-GFP (MBL), anti-HSC70 (Sigma Aldrich, St Louis, MO, USA), anti-Src (Millipore, Burlington, MA, USA) and rabbit polyclonal anti-β1 integrin (Millipore, Burlington, MA, USA), anti-EGFR (Cell Signaling, Danvers, MA, USA), anti-Erk1/2 (Cell Signaling, Danvers, MA, USA), anti-ezrin (Biotechne, Minneapolis, MN, USA), anti-FAK (Santa Cruz Biotechnology, Dallas, TX, USA), anti-phospho-EGFR (Y1068, Cell Signaling, Danvers, MA, USA), anti-phospho-EGFR (Y1137, Cell Signaling, Danvers, MA, USA), anti-phospho-Erk1/2 (T202, Y204, Cell Signaling, Danvers, MA, USA), anti-phospho-ezrin (Y478, Abcam, Cambridge, UK; noting the authors performed validation experiments to demonstrate the specificity of this antibody to ezrin; not shown), anti-phospho-FAK (Y397, Cell Signaling, Danvers, MA, USA) and anti-phospho-Src (Y418, Millipore, Burlington, MA, USA).

Techniques: Activity Assay, Stable Transfection, Expressing, Transfection, Immunostaining, Western Blot, Incubation, Immunoprecipitation, Negative Control

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: Mouse anti-CD29 (β 1 integrin) mAb (3S3) and FITC-conjugated mouse anti-β 1 integrin mAb (S3S) was from Serotec Inc. (Raleigh, NC).

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: Mouse anti-CD29 (β 1 integrin) mAb (3S3) and FITC-conjugated mouse anti-β 1 integrin mAb (S3S) was from Serotec Inc. (Raleigh, NC).

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: Mouse anti-CD29 (β 1 integrin) mAb (3S3) and FITC-conjugated mouse anti-β 1 integrin mAb (S3S) was from Serotec Inc. (Raleigh, NC).

Techniques: Inhibition, Expressing, Incubation, 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: Mouse anti-CD29 (β 1 integrin) mAb (3S3) and FITC-conjugated mouse anti-β 1 integrin mAb (S3S) was from Serotec Inc. (Raleigh, NC).

Techniques: Dominant Negative Mutation, Expressing, Stable Transfection, Transfection, Construct, 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: Mouse anti-CD29 (β 1 integrin) mAb (3S3) and FITC-conjugated mouse anti-β 1 integrin mAb (S3S) was from Serotec Inc. (Raleigh, NC).

Techniques: Expressing, Incubation, Flow Cytometry, Confocal Microscopy