Journal: Journal of Biological Chemistry

Article Title: Transactivation of the Epidermal Growth Factor Receptor Is Involved in 12-O-Tetradecanoylphorbol-13-acetate-induced Signal Transduction

doi: 10.1074/jbc.m107156200

Figure Lengend Snippet: FIG. 3. The neutralizing antibody of EGFR blocks TPA-in- duced ERK phosphorylation and AP-1 DNA binding activity. Panel A, effect of neutralizing antibodies for EGFR and HB-EGF on EGF- or TPA-induced EGFR phosphorylation. PC3 cells were cultured in a monolayer in 150-mm dishes until they reached 90% confluence and then starved in serum-free DMEM for 36 h. The cells were pre- treated with antibodies against 5 g/ml EGFR (EGFR) or 10 g/ml HB-EGF (HB-EGF) for 30 min and then treated with 600 ng/ml TPA or 10 ng/ml EGF for 3 min. The samples were immunoprecipitated by using anti-EGFR and probed with anti-phosphotyrosine (4G10) and anti-EGFR. Panel B, effect of a neutralizing antibody for EGFR on TPA- or EGF-induced ERK phosphorylation. PC3 cells (8 104/well) were cultured in a monolayer in six-well plates until they reach 90% conflu- ence, and then they were starved for 36 h in FBS-free and DMEM. The cells were pretreated with 0.5 g/ml antibody against EGFR for 30 min and then treated with 600 ng/ml TPA or 10 ng/ml EGF for 10 min. Samples were harvested with SDS sample buffer and analyzed by Western blotting with antibodies against nonphosphorylated or phos- phorylated ERKs. Panel C, effect on TPA-induced AP-1 DNA binding activity. PC3 cells were cultured in a monolayer in 10-cm dishes (1 106/dish) until they reached 90% confluence. Then, they were starved for 36 h in FBS-free and DMEM. The cells were pretreated with a 5 g/ml antibody against EGFR for 30 min followed by treatment with 600 ng/ml TPA for another 14 h. Sequence-specific AP-1 DNA binding activity was determined by gel shift analysis using a 32P-labeled oligo- nucleotide containing the AP-1 binding site.

Article Snippet: The antibodies used included rabbit polyclonal phosphorylated ERK antibodies (New England Biolabs, Beverly, MA), rabbit polyclonal EGFR (Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal phosphorylated tyrosine (4G10), mouse monoclonal EGFR (neutralizing, clone LA1, Upstate Biotechnologies, Lake Placid, NY), and mouse monoclonal heparin-binding EGF (HB-EGF) (neutralizing, R&D Systems Inc., Minneapolis, MN).

Techniques: Phospho-proteomics, Binding Assay, Activity Assay, Cell Culture, Immunoprecipitation, Western Blot, Sequencing, Gel Shift, Labeling

Journal: Journal of Biological Chemistry

Article Title: Transactivation of the Epidermal Growth Factor Receptor Is Involved in 12-O-Tetradecanoylphorbol-13-acetate-induced Signal Transduction

doi: 10.1074/jbc.m107156200

Figure Lengend Snippet: FIG. 4. A neutralizing antibody for HB-EGF blocks TPA- but not EGF-induced phosphorylation of ERKs. PC3 cells (8 104/ well) were cultured in a monolayer in six-well plates until they reached 90% confluence. Then they were starved for 12 h in 0.5% FBS and DMEM. The cells were pretreated with a 10 g/ml antibody against HB-EGF (HB-EGF) for 30 min and then treated with 600 ng/ml TPA or 10 ng/ml EGF for 10 min. Samples were harvested with SDS sample buffer and analyzed by Western blotting with antibodies against non- phosphorylated or phosphorylated ERKs.

Article Snippet: The antibodies used included rabbit polyclonal phosphorylated ERK antibodies (New England Biolabs, Beverly, MA), rabbit polyclonal EGFR (Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal phosphorylated tyrosine (4G10), mouse monoclonal EGFR (neutralizing, clone LA1, Upstate Biotechnologies, Lake Placid, NY), and mouse monoclonal heparin-binding EGF (HB-EGF) (neutralizing, R&D Systems Inc., Minneapolis, MN).

Techniques: Phospho-proteomics, Cell Culture, Western Blot

Journal: Nature Communications

Article Title: A critical role for NF2 and the Hippo pathway in branching morphogenesis

doi: 10.1038/ncomms12309

Figure Lengend Snippet: ( a – d ) Macroscopic view and periodic acid-Schiff (PAS) staining of YAP overexpressing and control kidneys at E18.5. Pregnant dams have been fed with doxycycline food (0.645 g kg −1 ) from E11 to E18.5. ( e – p ) E11.5 T-stage control kidney explants cultured ex vivo in presence of 1,500 ng ml −1 of doxycycline for 24 and 48 h shows the stereotypical branching morphogenesis pattern with an average of 22 ureteric tips in control kidneys after 48 h of cultures ( e – g ). ( h – j ) No branching is observed in Yap UB-OE kidneys as no new tip forms within 2 days in culture. ( k – m ) After 24 h of culture in normal media, the addition of doxycycline and induction of YAP transgene expression results in a complete block of further branching. ( n – p ) Branching morphogenesis blockage requires continuous YAP overexpression, as withdrawal of doxycycline from the medium after 24 h releases the inhibition, and new tips are formed. ( q ) Quantification of UB tip number in ex vivo explants experiments. Panels e , h , k and n represent the experimental flow of the ex vivo kidney cultures. The number of explants analysed for each genotype is indicated in the lower left corner of each panel. Scale bars represent 1 mm ( a , b ), 0.5 mm ( c , d ) and 250 μm ( f – p ).

Article Snippet: To induce YAP overexpression in kidney explants, doxycycline (R&D Systems, AF2028) was added to the culture medium at different concentrations (1,500, 150, 30 and 15 ng ml −1 ) from day 1 in culture, unless stated otherwise.

Techniques: Staining, Control, Cell Culture, Ex Vivo, Expressing, Blocking Assay, Over Expression, Inhibition

Journal: Nature Communications

Article Title: A critical role for NF2 and the Hippo pathway in branching morphogenesis

doi: 10.1038/ncomms12309

Figure Lengend Snippet: ( a – l ) Pregnant dams were fed with doxycycline food (0.645 g kg −1 ) from E11 until E13.5. YAP OE cells (red – HA positives) are only detected in triple transgenic ( Hoxb7:Cre tg/+ Rosa26-lox-STOP-lox-rtta-IRES-EGFP Yap Tg ) mice ( b – f ) and not in control animals ( a ). ( e , f ) Higher magnification views of c and d . ( g ) Quantification reveals that the percentage of YAP OE cells in the UB epithelium correlates with kidney size. ( h ) Diagram of denomination of tip (within 25 μm from the tip), tip adjacent (cells located 25–75 μm away from the tip) and trunk (cells located 75 μm and more from the tip) domains. ( i ) Quantification of the distribution of YAP OE cells in the UB compartments (tip, tip adjacent and trunk) in Yap UB-OE-Int mutants. Numbers in brackets represent the number of HA-positive cells and the total number of counted cells. ( j – l ) Immunostaining using anti-HA and ETV5 antibodies shows that the rare YAP OE cells present in the tip domain do not expressed the UB tip marker ETV5. Separate channels are shown in . ( m ) Quantification of UB tip numbers of E11.5 control and Yap UB-OE kidneys, after 48 h in culture exposed to different concentrations of doxycycline (15, 30 and 150 ng ml −1 ). Quantification was made on 8, 10 and 12 control explants and 7, 10 and 12 Yap UB-OE explants at 150, 30 and 15 ng ml −1 , respectively. ( n – q ) HA staining on kidney explant sections treated with 15 ng ml −1 of doxycycline (low enough to allow branching) reveals that cells with low YAP overexpression can contribute to UB tips. ( r ) Western blot analysis of kidney lysates confirms activation of YAP expression at different concentrations of doxycycline used in panels m – q . Scale bars represent 100 μm ( a – f , n – q ) and 50 μm ( j – l ).

Article Snippet: To induce YAP overexpression in kidney explants, doxycycline (R&D Systems, AF2028) was added to the culture medium at different concentrations (1,500, 150, 30 and 15 ng ml −1 ) from day 1 in culture, unless stated otherwise.

Techniques: Transgenic Assay, Control, Immunostaining, Marker, Staining, Over Expression, Western Blot, Activation Assay, Expressing

Journal: International journal of cancer

Article Title: Endothelin-1 stimulates motility of head and neck squamous carcinoma cells by promoting stromal-epithelial interactions.

doi: 10.1002/ijc.25968

Figure Lengend Snippet: Figure 5. Autocrine and paracrine effects of mitogenic peptides in HNSCC. ET-1 binds to its receptors on oral fibroblasts, activating ADAM17 and triggering the release from the cell surface of EGFR ligands such as HB-EGF, TGF-a and amphiregulin. Soluble EGFR ligands

Article Snippet: Where appropriate, the conditioned media was incubated for 30 min at 37 C with rotation following addition of neutralising antibodies to HB-EGF, TGF-a, amphiregulin (all R&D Systems, 10 ll/ml) or mouse IgG (Dako) before addition to transwells.

Techniques:

Journal: Molecular Vision

Article Title: Glucosamine inhibits epidermal growth factor-induced proliferation and cell-cycle progression in retinal pigment epithelial cells

doi:

Figure Lengend Snippet: Effects of glucosamine (GlcN) on transactivation of epidermal growth factor receptor (EGFR). Human retinal pigment epithelial cell line (ARPE-19) cells were treated with 2.5 mM or 5.0 mM GlcN, or 30 mM glucose for 24 h, stimulated with 10 ng/ml EGF, and stained with PE-conjugated anti-total-EGFR antibody and Alexa-Fluor-647-conjugated anti-p-EGFR antibody (Y845). The levels of phosphorylated ( A ) and total EGFR ( B ) were analyzed by flow cytometry. The data are representative of at least three independent experiments. Cells cultured under serum-free conditions were used as the control. C : western blot analysis of ARPE-19 cells cultured to 80% confluence; treated with 2 μg/ml tunicamycin, 30 mM glucose, or 5 mM GlcN in serum-free medium for a further 24 h; then stimulated with EGF for 5 min.

Article Snippet: The following primary antibodies were used: phycoerythrin (PE)-conjugated anti-total-EGFR (cells were not permeabilized for total EGFR detection) and Alexa-Fluor-647-conjugated anti-p-EGFR (Y845; BD PharMingen).

Techniques: Staining, Flow Cytometry, Cell Culture, Western Blot