Journal: The Journal of biological chemistry

Article Title: Enhanced tumorigenic behavior of glioblastoma cells expressing a truncated epidermal growth factor receptor is mediated through the Ras-Shc-Grb2 pathway.

doi: 10.1074/jbc.271.41.25639

Figure Lengend Snippet: FIG. 2. Interaction of phosphoproteins with Shc in U87MG cells expressing mutant EGFRs. A, all mutants analyzed contained a truncation of 801 base pairs in the EGFR extracellular domain. The D (DEGFR) mutant contained intact phosphorylation sites; DK contained a lysine to methionine point mutation at the ATP binding site (K721); DY1, DY2, DY3, DY4, and DY5 contained tyrosine to phenylalanine substitutions at known phosphorylation sites as indicated, where X denotes a mutated site. B, lysates prepared from EGF stimulated (1) and unstimulated (2) U87MG cells expressing truncated EGFR mu- tants were analyzed for the presence of the truncated receptors (a) and for the presence of tyrosine-phosphorylated proteins (b). Shc immuno- precipitates prepared from these cells were probed with an anti-phos- photyrosine antibody to detect the presence of phosphorylated mutant EGF receptors (c), and phosphorylated Shc (d). Shc precipitates were also analyzed for the presence of Grb2 associated with Shc (e)

Article Snippet: The monoclonal antibody used for specific precipitation of the truncated EGF receptor was raised against cells expressing the truncated EGFR and will be described elsewhere,2 and the antibody for precipitation of endogenous receptor was EGFR1 (Santa Cruz, sc-101).

Techniques: Expressing, Mutagenesis, Phospho-proteomics, Binding Assay

Journal: The Journal of biological chemistry

Article Title: Enhanced tumorigenic behavior of glioblastoma cells expressing a truncated epidermal growth factor receptor is mediated through the Ras-Shc-Grb2 pathway.

doi: 10.1074/jbc.271.41.25639

Figure Lengend Snippet: FIG. 3. Interaction of Shc and Grb2 with truncated EGFR mu- tants. Lysates were prepared from U87MG cells expressing truncated receptor mutants, and U87MG cells expressing wild-type EGFR (U87MG.wtEGFR), which had been pretreated with EGF (1) or were untreated (2). Total lysate was analyzed to demonstrate the difference in phosphorylation state of the receptor mutants (b). Truncated EGFR was precipitated from equal amounts of total protein using an antibody specific for the mutant receptor, except in the case of the U87MG.wtEGFR cells where an antibody recognizing the full-length receptor was used. Immunoprecipitates were analyzed for EGFR (a) Shc (c) and Grb2 (d) by Western blotting.

Article Snippet: The monoclonal antibody used for specific precipitation of the truncated EGF receptor was raised against cells expressing the truncated EGFR and will be described elsewhere,2 and the antibody for precipitation of endogenous receptor was EGFR1 (Santa Cruz, sc-101).

Techniques: Expressing, Phospho-proteomics, Mutagenesis, Western Blot

Journal: Cellular signalling

Article Title: Features of the reversible sensitivity-resistance transition in PI3K/PTEN/AKT signalling network after HER2 inhibition.

doi: 10.1016/j.cellsig.2011.09.030

Figure Lengend Snippet: Fig. 2. Dose response and sensitivity properties of the PI3K/PTEN/AKT signalling network. (A) pHER2 dose dependence for heregulin-β (HRG) concentration (solid line), pAKT dose dependence for HRG in the absence (dashed line) and presence of PTEN inhibitor, 50 nM bpV(pic) (dotted line). (B) pHER2 and pAKT dose dependencies for pertuzumab (solid and dashed lines, respectively) at 95 nM and 1 μM of HER2 concentration (thick and thin lines, respectively). Dotted line — pAKT dose dependence at three-fold increase in activity of CK2/GSK3β reaction of PTEN phosphorylation. Points on thick lines — experimental data (see Fig. 3 in [33]). (C) pHER2 and pAKT dose dependencies for HER2 concentration in the absence (thick solid and dashed lines, respectively) and presence of 100 nM pertuzumab (thin solid and dashed lines, respectively). (D) The dependence of sensitivities of the whole signalling network, SSN (solid line), receptor subsystem SRSS (dotted line), and signalling transaction subsystem SSTS (dashed line) on pertuzumab concentration. (E) Western blot analysis of the dose dependence of pHER2 (left) and pAKT (right) to HRG concentration (0 nM (control), 0.01 nM, 0.1 nM, 1 nM and 10 nM) at 5 and 30 min.

Article Snippet: Cells were treatedwith UCN-01 (protein kinase inhibitor; Calbiochem#539644; final concentration of 1 μM), LY294002 (PI3 kinase inhibitor; Calbiochem#440204; final concentration 20 μM), pertuzumab (HER2 inhibitor; final concentration 100 nM) and stimulation by heregulin (R&D Systems; 396-HB-CF) was at final concentration of 1 nM.

Techniques: Concentration Assay, Activity Assay, Phospho-proteomics, Western Blot, Control

Journal: Cellular signalling

Article Title: Features of the reversible sensitivity-resistance transition in PI3K/PTEN/AKT signalling network after HER2 inhibition.

doi: 10.1016/j.cellsig.2011.09.030

Figure Lengend Snippet: Fig. 6. (A) Western blot analysis of the inhibition effect on pAKT of varying concentrations of PTEN inhibitor, bpV(pic) (5 nM, 10 nM, 25 nM, 50 nM, 100 nM) at 1 nM heregulin, HRG in PE04 cells. (B) Theoretical pAKT dose dependence on PTEN concentration at saturated HRG signal (thin line) and at HER2 inhibition by pertuzumab (thick line). Squares — ex- perimental data on the dependence of pAKT concentration on PTEN expression level in 13 ovarian cancer lines (in relative units). Circles — experimental data on the dependence of pAKT concentration on PTEN expression for basal-like breast carcinoma (in relative units) [28]. (C) Experimental data (mean±S.D., n=3) on the effects of combinations of PDK1 in- hibition by 7.5 μM UCN-01, HER2 inhibition by 100 nM pertuzumab (2C4) and PTEN inhi- bition by 50 μM bpV(pic).

Article Snippet: Cells were treatedwith UCN-01 (protein kinase inhibitor; Calbiochem#539644; final concentration of 1 μM), LY294002 (PI3 kinase inhibitor; Calbiochem#440204; final concentration 20 μM), pertuzumab (HER2 inhibitor; final concentration 100 nM) and stimulation by heregulin (R&D Systems; 396-HB-CF) was at final concentration of 1 nM.

Techniques: Western Blot, Inhibition, Concentration Assay, Expressing

Journal: Cellular signalling

Article Title: A novel epidermal growth factor receptor-signaling platform and its targeted translation in pancreatic cancer.

doi: 10.1016/j.cellsig.2013.08.008

Figure Lengend Snippet: Fig. 2. (A) Tamiflu and anti-Neu1 neutralizing antibody inhibit EGF induced EGFR phosphorylation (pEGFR) in 3T3–hEGFR cells. Cells were grown overnight on glass coverslips in a 24-well tissue culture plate at 37 °C for 24 h or until they reached ~70% confluence. Cells were stimulated with 30 ng/mL EGF for 5 min, pretreated with 200 μM Tamiflu for 30 min followed by 30 ng/mL EGF for 5 min, or pretreated with 100 μg/mL anti-Neu1, -2, -3, or -4 neutralizing antibodies for 30 min followed by 30 ng/mL EGF for 5 min. Cells were left untreated as no ligand controls. Cells were fixed with 4% paraformaldehyde, permeabilized with Triton X, and blocked with 4% bovine serum albumin (BSA) in 0.1% Tween–Tris buffered saline (TBS) for 20 min on ice. Cells were immunostained with rabbit anti-human pEGFR for 60 min at 37 °C, followed by AlexaFluor 594 goat anti-rabbit secondary antibody for 60 min at 37 °C. Control group had only secondary antibodies with no other treatment. Stained cells were visualized by epi-fluorescence microscopy with a 40× objective. Quantitative analysis was done by assessing the density of cell staining corrected for background in each panel using Corel Photo Paint 8.0 software. Each bar in the figure represents the mean corrected density of culture cell staining ± SEM for equal cell density (5 × 105 cells) within the respective images. The data are a representation of one out of three independent experiments showing similar results. (B) Western blot analyses of Tamiflu and anti-Neu1 neutralizing antibody inhibition of EGF-induced pEGFR in 3T3–hEGFR cell lysates. Cell were treated 30 ng/mL EGF for 5 min or pretreated with either 400 μM Tamiflu or 100 μg/mL anti-Neu1 neutralizing antibody for 30 min or left untreated as control (media). Cells were pelleted, lysed in lysis buffer and the cell lysates were resolved by SDS-PAGE. The blot was probed with 0.14 μg/mL rabbit anti-human pEGFR antibody overnight at 4 °C followed by 40 ng/mL horse radish peroxidase-labeled goat anti- rabbit antibody for 75 min at 20 °C and Western Lightning Chemiluminescence Reagent Plus for 5 min. NIH3T3 cells served as EGFR negative control. After development, blots were stripped and re-probed with rabbit anti-human pan-EGFR as a loading control. The data are a representation of one out of three independent experiments showing similar results. (C) A Western blot was performed as described in (B) above, but 3T3–hEGFR cells were stimulated with 30 ng/mL EGF for 10 min. (D) Immunoprecipitation of EGFR and Western blot analyses of biotinylated cell surface of 3T3–hEGFR cells in the presence of Tamiflu, anti-Neu1 neutralizing antibody and specific MMP-9 inhibitor. Cells were left untreated (control), stimulated with 30 ng/mL EGF for 5 min, or pretreated with 400 μM Tamiflu, 100 μg/mL anti-Neu1 neutralizing antibody or 50 μg/mL MMP-9 inhibitor for 30 min followed by 30 ng/mL EGF stimulation for 5 min. Cells were biotinylated with NHS-SS-biotin on ice for 30 min, extensively washed, pelleted and lysed in lysis buffer. The EGFR in the cell lysates was immunoprecipitated with 1 μg of goat anti-EGFR antibody overnight at 4 °C. Immunocomplexes were isolated using protein G magnetic beads, resolved by SDS-PAGE and the blot probed with streptavidin–HRP followed by Western Lightning Chemiluminescence Reagent Plus. NIH3T3 and 3T3–hEGFR cells that were not immunoprecipitated (no IP Ab) served as negative controls. The data are a representation of one out of two independent experiments showing similar results. (E) Maackia amurensis lectin 2 (MAL-2) dose-dependently inhibits EGF-induced pEGFR in human skin epidermoid carcinoma A431 cell line. A431 cells were starved in serum free media for 24 h. The cells were pretreated with MAL-2, Sambucus nigra lectin (SNA), peanut agglutinin (PNA) and succinylated wheat germ agglutinin (sWGA) lectins at indicated doses for 30 min followed by 30 ng/mL EGF for 5 min or left unstimulated as control. Cells were fixed with 4% paraformaldehyde, permeabilized with Triton X100 and immunostained with mouse anti-pEGFR followed by AlexaFluor 488 rabbit anti-mouse IgG. Stained cells were visualized by epi- fluorescence microscopy using a 40× objective. Quantitative analysis was done by assessing the density of cell staining corrected for background in each panel using Corel Photo Paint 8.0 software. Each bar in the graphs represents the mean corrected density of staining ± S.E. (error bars, n = 4) for equal cell density (5 × 105 cells) within the respective images. Results were compared by a one-way ANOVA at 95% confidence using Bonferroni's multiple comparison test. The data are a representation of one of five independent experiments showing similar results.

Article Snippet: Preclinical molecular-targeting studies focused on inhibiting Neu1 as the key central enzyme within this novel EGFR signaling paradigm provide the proof-of-evidence for an effective Tamiflu monotherapy in the treatment of human pancreatic cancer growth and metastatic spread in heterotopic xenograft of tumors growing in RAG2−/−xCγ−/− doublemutant mice.

Techniques: Phospho-proteomics, Saline, Control, Staining, Microscopy, Software, Western Blot, Inhibition, Lysis, SDS Page, Labeling, Negative Control, Immunoprecipitation, Isolation, Magnetic Beads, Comparison

Journal: Cellular signalling

Article Title: A novel epidermal growth factor receptor-signaling platform and its targeted translation in pancreatic cancer.

doi: 10.1016/j.cellsig.2013.08.008

Figure Lengend Snippet: Fig. 4. (A) Neu1 co-immunoprecipitates with EGFR. 3T3–hEGFR cells were treated with 30 ng/mL EGF for 5 min, or pretreated with 400 μM Tamiflu for 30 min followed by 30 ng/mL EGF for 5 min or left untreated as control (media). Cells were pelleted, lysed in lysis buffer and the protein lysates were immunoprecipitated with 1 μg of goat anti-EGFR antibody overnight at 4 °C. Immunocomplexes were isolated using protein G magnetic beads, resolved by SDS-PAGE and the blot probed with rabbit anti-Neu1 antibody overnight at 4 °C followed by HRP- conjugated goat anti-rabbit HRP-conjugated secondary antibody for 75 min at 20 °C and Western Lightning Chemiluminescence Reagent Plus. NIH3T3 and 3T3–hEGFR cells not immunoprecipitated (no IP Ab) served as negative controls. The data are a representation of one out of four independent experiments showing similar results. (B) Neu-2 and -3 do not co-immunoprecipitate with EGFR. 3T3–hEGFR cells were used as described in (A). (C) Western blot of unstimulated 3T3–hEGFR cells was run simultaneously with the immunoprecipi- tation to detect levels of Neu-2, -3, and -4 proteins in the same cell lysates. The data are a representation of one out of three independent experiments showing similar results.

Article Snippet: Preclinical molecular-targeting studies focused on inhibiting Neu1 as the key central enzyme within this novel EGFR signaling paradigm provide the proof-of-evidence for an effective Tamiflu monotherapy in the treatment of human pancreatic cancer growth and metastatic spread in heterotopic xenograft of tumors growing in RAG2−/−xCγ−/− doublemutant mice.

Techniques: Control, Lysis, Immunoprecipitation, Isolation, Magnetic Beads, SDS Page, Western Blot