Journal: Cancer research

Article Title: E-Cadherin Cell-Cell Adhesion in Ewing Tumor Cells Mediates Suppression of Anoikis through Activation of the ErbB4 Tyrosine Kinase

doi: 10.1158/0008-5472.CAN-06-3259

Figure Lengend Snippet: Activation of the ErbB4 RTK in ET spheroids. A, the R&D Systems Human Phospho-RTK Antibody Proteome Profiler Array system was used to screen for activation of specific tyrosine kinases in ET spheroids. Lysates from TC32 and TC71 monolayer and spheroid cultures were incubated with membranes arrayed with antibodies from 42 different tyrosine kinases (http://www.rndsystems.com/). Membranes were then washed and incubated with anti–phosphotyrosine-HRP antibodies followed by enhanced chemiluminescence detection to identify activated tyrosine kinases. P-Tyr, phospho-tyrosine control spots. B, top, lysates from TC32 and TC71 monolayer and spheroid cultures were subjected to immunoprecipitation (IP) with 4G10 anti-phosphotyrosine antibodies and then immunoblotted (IB) with an anti-ErbB4 antibody. The positions of 180- and 80-kDa tyrosine phosphorylated species are indicated. n.s., nonspecific band. Left, lysates from TC32 and TC71 monolayer and spheroid cultures were subjected to immunoprecipitation with antibodies to total ErbB4 followed by immunoblotting with anti-phosphotyrosine antibodies. Right, the blot was then reprobed with an anti-ErbB4 antibody. The positions of 180- and 80-kDa tyrosine phosphorylated species are indicated. C, TC32 and TC71 cells were grown as monolayers or spheroids as indicated in 10% serum–containing media for 48 h. Cells were then lysed and subjected to immunoblotting with antibodies to activated ErbB4 (phospho-Tyr1188). Detection of β-actin was used as a loading control. The positions of 180- and 80-kDa tyrosine phosphorylated species are indicated. D, ErbB4 and E-cadherin expression was analyzed by immunohistochemistry from sections from primary ET. Sections were subjected to standard H&E staining, as well as immunohistochemistry with anti-ErbB4 (ErbB4), anti–E-cadherin, and control IgG antibodies. Magnification, ×400.

Article Snippet: Standard Western blot analysis was done with antibodies to poly(ADP-ribose) polymerase, phospho-Akt Ser 473 , total Akt, phospho–mitogen-activated protein kinase/ERK kinase (MEK) 1/2 Ser 217/221 (Cell Signaling, Beverly, MA); β-actin, ErbB4, ErbB2 (Santa Cruz Biotechnology, Santa Cruz, CA); phospho-ErbB4 (Tyr 1188 ), phospho-ErbB2 (Tyr 1112 ) (Orbigen, San Diego, CA); E-cadherin, Rac1 (BD Transduction Laboratories, San Diego, CA); and phosphotyrosine (4G10 from Upstate, Lake Placid, NY).

Techniques: Activation Assay, Incubation, Immunoprecipitation, Western Blot, Expressing, Immunohistochemistry, Staining

Journal: Cancer research

Article Title: E-Cadherin Cell-Cell Adhesion in Ewing Tumor Cells Mediates Suppression of Anoikis through Activation of the ErbB4 Tyrosine Kinase

doi: 10.1158/0008-5472.CAN-06-3259

Figure Lengend Snippet: ErbB4 RNA interference blocks Akt activation and restores chemosensitivity of ET spheroids. A, TC32 and TC71 cell lines that were either nontransduced or stably expressing DN-Ecad or its vector alone or Ecad or its vector alone were grown as monolayers or spheroids as indicated in 10% serum–containing media for 48 h. Cells were then lysed and subjected to immunoblotting with antibodies to activated ErbB4 (phospho-Tyr1188; p-ErbB4) or total ErbB4. B, ErbB4 knockdown with RNA interference blocks Akt activation. TC32 spheroids grown in media containing 10% serum were treated with two ErbB4-specific siRNA pools (siRNA1 or siRNA2), as described in Materials and Methods, or a scrambled siRNA control. Lysates were then subjected to immunoblotting with antibodies to total ErbB4, phospho-Akt (pAKT), phospho-MEK (pMEK), or β-actin as a loading control. C, phase-contrast photomicrographs of TC32 spheroids after pretreatment of cells with an ErbB4-specific siRNA (siRNA1) or a scrambled siRNA control. Magnification, ×40.D, reduction of ErbB4 by RNA interference restores chemosensitivity of ET spheroids. TC32 spheroids grown in media containing 10% serum were treated with ErbB4-specific siRNAs or a scrambled control as above. Cells were then treated with vehicle control, 50 µmol/L etoposide, or 10 µmol/L doxorubicin for 24 h after siRNA transfection. Caspase-3 activity was then assayed as above with normalization to a value of 1.0 arbitrary unit for nontreated cells. Statistical analysis of data from at least three separate experiments was done using Student's t test. Horizontal lines, P values comparing scrambled siRNA control with ErbB4 siRNA1 cells in the presence of etoposide (P < 0.004) or doxorubicin (P < 0.009), or comparing scrambled siRNA control with ErbB4 siRNA2 in the presence of etoposide (P < 0.0006) or doxorubicin (P < 0.003).

Article Snippet: Standard Western blot analysis was done with antibodies to poly(ADP-ribose) polymerase, phospho-Akt Ser 473 , total Akt, phospho–mitogen-activated protein kinase/ERK kinase (MEK) 1/2 Ser 217/221 (Cell Signaling, Beverly, MA); β-actin, ErbB4, ErbB2 (Santa Cruz Biotechnology, Santa Cruz, CA); phospho-ErbB4 (Tyr 1188 ), phospho-ErbB2 (Tyr 1112 ) (Orbigen, San Diego, CA); E-cadherin, Rac1 (BD Transduction Laboratories, San Diego, CA); and phosphotyrosine (4G10 from Upstate, Lake Placid, NY).

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

Journal: Translational Psychiatry

Article Title: Clozapine-dependent inhibition of EGF/neuregulin receptor (ErbB) kinases

doi: 10.1038/s41398-019-0519-1

Figure Lengend Snippet: Inhibition of EGF-induced ErbB1 phosphorylation by antipsychotic compounds in U87MG cells ( a ) and cultured cortical neurons ( b ) was determined by immunoblotting for phosphorylated (P-ErbB1) and total ErbB1 (ErbB1). c The inhibition of neuregulin-1 (NRG)-induced ErbB4 phosphorylation by antipsychotic compounds in cultured cortical neurons was similarly determined by immunoblotting. Typical immunoblots for phosphorylated and total ErbB4 are shown. Bar represents the ratio to the EGF-induced or neuregulin-1-induced phosphorylation levels (100% maximum) obtained in the absence of antipsychotic compounds (mean ± SE, n = 3 sister cultures). Horizontal broken lines mark the averaged basal phosphorylation level of ErbB1 (None; 0.6 ± 0.12% for U87MG; 3.5 ± 0.7% for cortical neurons) and that of ErbB4 in control cultures (None; 54.0 ± 4.6%). The abbreviations of the antipsychotic compounds are same as indicated in Fig. . * P < 0.05 and ** P < 0.01 vs. positive control cultures (100%; DMSO), †† P < 0.01 and ††† P < 0.001 vs. negative control cultures (None), one-way ANOVA followed by Tukey’s test. β = 0.104 and η 2 = 0.710 for a , β = 0.051 and η 2 = 0.750 for b , β < 0.001 and η 2 = 0.894 for c . Brown–Forsythe test suggested the homogeneity of data variance; P = 0.515 for a , 0.829 for b , and 0.844 for c . NRG; neuregulin-1

Article Snippet: Additionally, the following primary antibodies were used; anti-phospho-Tyr1139-ErbB2 (1:1000, Cat#1991, Eptomics, Curlingame, CA USA), anti-ErbB2 (1:1000, Cat#2064, Eptomics Abcam), anti-phospho-Tyr1328-ErbB3 (1:1000, Cat#135654, Santa Cruz Biotechnology, Texas, USA), phospho-Tyr1173-ErbB1 (1:1000, Cat#sc-12351, Santa Cruz), anti-ErbB3 (1:1000, Cat#1186, Eptomics), anti-phospho-Tyr1056-ErbB4 (1:1000, Cat#13094, Bioss, Boston, USA), anti-phospho-Tyr1242-ErbB4 (1:1000, Cat#12727, Signalway Antibody, Maryland, USA), and anti-ErbB4 (1:1000, Cat#2218, Eptomics) , .

Techniques: Inhibition, Cell Culture, Western Blot, Positive Control, Negative Control

Journal: Translational Psychiatry

Article Title: Clozapine-dependent inhibition of EGF/neuregulin receptor (ErbB) kinases

doi: 10.1038/s41398-019-0519-1

Figure Lengend Snippet: a Phosphorylation levels of ErbB1 in U87MG cells treated with 0–100 µM clozapine (CLZ) are presented. b Phosphorylation levels of ErbB4 in cultured cortical neurons pretreated with 0–100 µM clozapine are presented. The graph reveals % ratio to the EGF-induced or neuregulin-1-induced phosphorylation levels (100% maximum) (mean ± SE, n = 3 sister cultures). Horizontal broken lines mark the averaged basal phosphorylation level of ErbB1 (None; 1.1 ± 0.7%) or that of ErbB4 in untreated cultures (None; 27.0 ± 3.3%). * P < 0.05, ** P < 0.01, and *** P < 0.001 vs. control cultures (0 μM clozapine), † P < 0.05, †† P < 0.01, and ††† P < 0.001 vs. negative control cultures (None), ## P < 0.01 between adjacent concentrations, one-way ANOVA followed by Tukey’s test. β = 0.023 and η 2 = 0.781 for a , β < 0.001 and η 2 = 0.936 for b . Brown–Forsythe test suggested the homogeneity of data variance; P = 0.829 for a and 0.788 for b . NRG1; neuregulin-1

Article Snippet: Additionally, the following primary antibodies were used; anti-phospho-Tyr1139-ErbB2 (1:1000, Cat#1991, Eptomics, Curlingame, CA USA), anti-ErbB2 (1:1000, Cat#2064, Eptomics Abcam), anti-phospho-Tyr1328-ErbB3 (1:1000, Cat#135654, Santa Cruz Biotechnology, Texas, USA), phospho-Tyr1173-ErbB1 (1:1000, Cat#sc-12351, Santa Cruz), anti-ErbB3 (1:1000, Cat#1186, Eptomics), anti-phospho-Tyr1056-ErbB4 (1:1000, Cat#13094, Bioss, Boston, USA), anti-phospho-Tyr1242-ErbB4 (1:1000, Cat#12727, Signalway Antibody, Maryland, USA), and anti-ErbB4 (1:1000, Cat#2218, Eptomics) , .

Techniques: Cell Culture, Negative Control

Journal: Translational Psychiatry

Article Title: Clozapine-dependent inhibition of EGF/neuregulin receptor (ErbB) kinases

doi: 10.1038/s41398-019-0519-1

Figure Lengend Snippet: Recombinant intracellular domain proteins of ErbB1, B2, and B4 were obtained from the commercial sources and employed in the present in vitro kinase assay using the substrate of Tyr-Glu heteropolymers. The in vitro kinase assay was performed in the presence of 0–100 µM clozapine in the non-reducing condition (see Materials and Methods), and the product of phosphorylated tyrosine was quantitated by enzyme immunoassay with the peroxidase-conjugated anti-phosphotyrosine antibody. The graph reveals % inhibition of the ErbB kinase activity (mean ± SE, n = 5 assay wells), compared to the basal enzyme activity (0% inhibition). * P < 0.05, ** P < 0.01, and *** P < 0.001 vs. control data (0 μM clozapine), ## P < 0.01 between adjacent concentrations, one-way ANOVA followed by Tukey’s test. β = 0.003 and η 2 = 0.662 for ErbB1 kinase, β < 0.001 and η 2 = 0.684 for ErbB2 kinase, β < 0.001 and η 2 = 0.774 for ErbB4 kinase. Brown–Forsythe test suggested the homogeneity of data variance; P = 0.780 for ErbB1 kinase, 0.768 for ErbB2 kinase, and 0.650 for ErbB4 kinase. NRG; neuregulin-1

Article Snippet: Additionally, the following primary antibodies were used; anti-phospho-Tyr1139-ErbB2 (1:1000, Cat#1991, Eptomics, Curlingame, CA USA), anti-ErbB2 (1:1000, Cat#2064, Eptomics Abcam), anti-phospho-Tyr1328-ErbB3 (1:1000, Cat#135654, Santa Cruz Biotechnology, Texas, USA), phospho-Tyr1173-ErbB1 (1:1000, Cat#sc-12351, Santa Cruz), anti-ErbB3 (1:1000, Cat#1186, Eptomics), anti-phospho-Tyr1056-ErbB4 (1:1000, Cat#13094, Bioss, Boston, USA), anti-phospho-Tyr1242-ErbB4 (1:1000, Cat#12727, Signalway Antibody, Maryland, USA), and anti-ErbB4 (1:1000, Cat#2218, Eptomics) , .

Techniques: Recombinant, In Vitro, Kinase Assay, Enzyme-linked Immunosorbent Assay, Inhibition, Activity Assay

Journal: Translational Psychiatry

Article Title: Clozapine-dependent inhibition of EGF/neuregulin receptor (ErbB) kinases

doi: 10.1038/s41398-019-0519-1

Figure Lengend Snippet: Clozapine (20 mg/kg) or saline was intraperitoneally injected to adult rats and maintained for 1 h. Frontal cortex ( a ) and hippocampus ( b ) were taken and subjected to immunoblotting for phosphorylated Try1173 of ErbB1 (P-ErbB1) and Try1284 of ErbB4 (P-ErbB4) and for total ErbB1 and ErbB4. Typical immunoblots for phosphorylated and total ErbB1 and ErbB4 are shown as examples. The ratio of P-ErbB1 to total ErbB1 and that of P-ErbB4 to total ErbB4 in the saline-injected control rats are set to 100% (mean ± SE, n = 4 rats for each group). In a β = 0.856 and η 2 = 0.154 for P-ErbB1/ErbB1, β = 0.341 and η 2 = 0.808 for P-ErbB4/ErbB4. In b β = 0.575 and η 2 = 0.425 for P-ErbB1/ErbB1, β = 0.280 and η 2 = 0.902 for P-ErbB4/ErbB4. Brown–Forsythe test suggested the homogeneity of data variance; P = 0.936–0.603 for a , and P = 0.427–0.414 for b

Article Snippet: Additionally, the following primary antibodies were used; anti-phospho-Tyr1139-ErbB2 (1:1000, Cat#1991, Eptomics, Curlingame, CA USA), anti-ErbB2 (1:1000, Cat#2064, Eptomics Abcam), anti-phospho-Tyr1328-ErbB3 (1:1000, Cat#135654, Santa Cruz Biotechnology, Texas, USA), phospho-Tyr1173-ErbB1 (1:1000, Cat#sc-12351, Santa Cruz), anti-ErbB3 (1:1000, Cat#1186, Eptomics), anti-phospho-Tyr1056-ErbB4 (1:1000, Cat#13094, Bioss, Boston, USA), anti-phospho-Tyr1242-ErbB4 (1:1000, Cat#12727, Signalway Antibody, Maryland, USA), and anti-ErbB4 (1:1000, Cat#2218, Eptomics) , .

Techniques: Injection, Western Blot