Phospho erbb3

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).

Antibodies for western blotting analyses were purchased from Cell Signaling Technology (Boston, MA, USA). Anti-ErbB3 and anti-GAPDH were obtained from Santa Cruz Biotechnology (Dallas, TX, USA) [13 (link),17 (link)]. Anti-rabbit and anti-mouse were purchased from AbCam (Cambridge, UK). Vemurafenib and trametinib were obtained from Selleck Chemicals (Houston, TX, USA). TaqMan probes for ErbB3, HRG, FOXD3 and housekeeping gene GAPDH were purchased from Applied Biosystems (Foster City, CA, USA). Actinomycin D was purchased from Sigma (St. Louis, MO, USA). The anti-NRG (blocking peptide) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Immunofluorescence primary antibody phospho-ErbB3 was obtained from Santa Cruz Biotechnology.