Erbb3

Whole-cell lysates were prepared in lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% IGEPAL CA-630) with protease inhibitors (Complete EDTA-free protease inhibitor tablets, Roche Applied Science, Inc., Indianapolis, IN, USA, cat. no. 11836170001) and phosphatase inhibitors (PhosSTOP, Roche Applied Science, Inc., cat. no. 4906845001). Samples were clarified by centrifugation in a microfuge at 4 degrees for 10 min. Samples were run on SDS-PAGE gels under reducing conditions and transferred to nitrocellulose. Blots were probed for ERBB1 (Santa Cruz, Inc., cat. no. sc-03), ERBB2 (Lab Vision, Inc., cat. no. MS-599-P1), ERBB3 (Santa Cruz, Inc., cat. no. sc-285), ERBB4 (Cell Signaling Technology, Inc., cat. no. 4795), RET (R&D Systems, Inc., cat. no. AF482), NRG1α1 (R&D Systems, Inc., cat. no. AF296NA), NRG1β1 (R&D Systems, Inc., cat. no. AF396NA), phospho-AKT-S473 (Cell Signaling Technology, Inc., cat. no. 9271), phospho-P42/44 MAPK (Cell Signaling Technology, Inc., cat. no. 9101) and alpha-tubulin (Biogenex, Inc., Fremont, CA, USA, cat. no. MU121-UC). Secondary antibodies labeled with IRDye 800CW or 680LT were from Li-Cor Biosciences, Inc. (Lincoln, NB, USA). Blots were imaged on Odyssey Classic Quantitative Fluorescence Imaging System, Model 9120, also from Li-Cor Biosciences.

NVP-AEW541 and NVP-BEZ235 were gifts from Novartis (Basel, Switzerland). Lapatinib, KU0063794 and LY294002 were from Cayman Chemical (Michigan, USA). Cisplatin was purchased from Sigma-Aldrich (Steinheim, Germany). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Serva (Heidelberg, Germany). Propidium iodide was from PromoCell (Heidelberg, Germany). Roswell Park Memorial Institute (RPMI) media 1640, Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin [10,000 U/ml; 10 mg/ml] and trypsin-EDTA (0.05% trypsin, 0.02% EDTA in PBS) were purchased from PAN Biotech (Aidenbach, Germany). Primary antibodies were purchased from R&D Systems (Wiesbaden, Germany) (pIGF1R, IGF1R, p-EGFR, EGFR, p-ErbB2, ErbB2, p-ErbB3, ErbB3) or Santa Cruz Biotechnology (Heidelberg, Germany) (p-Akt, Akt, β-Actin, PARP). HRP-conjugated secondary antibodies were from R&D Systems. All other reagents and chemicals were from VWR BDH PROLABO (Darmstadt, Germany).

Expression of ERBB1-4 receptor proteins was determined by IHC using commercial monoclonal antibodies EGFR (1:50, Santa Cruz), ERBB-2 (1:50, Santa Cruz), ERBB-3 (1:50, Santa Cruz) and ERBB-4 (1:50, Santa Cruz). The proliferation of tumor cells was detected by ki-67 IHC staining. Formalin-fixed and paraffin-embedded sections, 4 μm thick, with representative tumor tissue, were incubated with primary antibodies after antigen retrieval by pressure autoclaving. An automatized histostainer was used for the immunohistochemcial procedures (Dako Autostainer, Denmark). For visualization of immunoreactivity, DAKO EnVision system was used with diaminobenzidin as chromogene. Sections were counterstained with haematoxylin. Positive controls were included in each staining run.
The immunoreactivity was assessed by means of intensity and percentage of immunoreactive tumor cells. Intensity was recorded as 0 (no reaction) to 3 (strong reaction). Fraction of immunoreactive tumor cells was recorded as 0 (no positive cells), 1 (<10% positive cells), 2 (10-50% positive cells), or 3 (>50% positive cells). A staining index was calculated as the product of intensity and fraction of positive tumor cells [15 ].

Rat tumor tissues, cultured cells and acetone-precipitated supernatants were lysed in a modified radioimmunoprecipitation assay protein extraction buffer as described previously [4 (link)]. Each sample was applied equally to 8–15% polyacrylamide gels and transblotted to polyvinylidene difluoride membranes (GE healthcare, Uppsala, Sweden). After blocking, primary-antibodies to AKT1 (1:2000, 2938), phospho-AKT1 (1:1000, 9271), Caspase-3 (1:1000, 9665), ERK1/2 (1:1000, 4695), phospho-ERK1/2 (1:1000, 4370), LC3B (1:1000, 3868), mTOR (1:1000, 2972), phospho-mTOR (1:1000, 2971), PI3K p110α (1:1000, 249), phospho-PI3K p85/p55 (1:500, 4228) (Cell Signaling Technology, Inc., Danvers, MA, USA), β-actin (1:1000, sc-47778), EGFR (1:250, sc-03), ErbB2 (1:1000, sc-284), ErbB3 (1:1000, sc-285), phospho-ErbB2 (1:500, sc-81508), phospho-ErbB3 (1:250, sc-135654), HIF-1α (1:1000, sc-53546), GAPDH (1:2000, sc-20357) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), phospho-EGFR (1:1000, 44-790G; Thermo Fisher Scientific), αSMA (1:2000, ab32575; abcam), E-cadherin (1:2000, 610181; BD Biosciences) and HCaRG (1:2000) were incubated overnight, followed by incubation with secondary horseradish-peroxidase conjugated-antibodies (Santa Cruz Biotechnology, Inc.) for 60 minutes. Immunocomplexes were detected by enhanced chemiluminescence (PerkinElmer Life Sciences).

Animals were fixed and stained as previously described [26 ,46 (link)]. The primary antibodies used in this study include: Sox10—1:5,000 [47 ], Acetylated Tubulin 1:10,000 (Sigma), βIII Tubulin 1:1,000 (Covance), S100 1:1,000 (Dako) [48 (link)], ErbB3 1:200 (Santa Cruz) and Isl1 1:100 (Developmental Studies Hybridoma Bank). The secondary antibodies used include Alexa antibodies (Invitrogen) (1:600); goat anti-rabbit 568, goat anti-mouse 568, goat anti-rabbit 647 and goat anti-mouse 647. After staining, zebrafish animals were stored in 50% glycerol/50% 1X PBS until imaged when they were mounted under a bridged coverslip.