Anti-ErbB3 antibody RTJ2 (Abcam); anti-erbB-3/HER-3 antibody, clone 2F12 (Millipore); mouse anti-human c-erbB-3 (BD Biosciences); phospho-HER3/ErbB3 (Tyr1289) (21D3) rabbit mAb, anti-b-actin, anti-b-tubulin (Santa Cruz); anti-GAPDH, anti-AKT, anti-pAKT, anti-pERK1/2, anti-p44/42 MAP kinase (Cell Signaling Technology) were used in the study. Secondary antibodies were purchased from Jackson ImmunoResearch or Invitrogen. DNaseI-RNase free and Ribonucleoside-vanadyl complex were from NEB. Human tumor cell lines used (BT-549, BT-474, Hs587T, MCF-7, MCF10A, SKBR-3, MDA-MB-231, MDA-MB-468 and HEK293) were from ATCC and grown in RPMI or D-MEM containing 10% FBS. The HER3 neutralizing antibodes (HER3Mabs) were produced in our laboratory and described previously [20 (link), 36 (link)]. Breefly, the HER3 neutralizing antibodies HER3Mabs are a monoclonal antibodies with backbone of IgG1. HER3Mabs were expressed in HEK293 freestyle cells (Life Technologies) and purified to above 95% purity using protein A/G affinity chromatography. Antibody purity was verified by protein gel electrophoresis and antibody binding was confirmed by ELISA binding assays, flow cytometry analysis. The ability to inhibit HER3 phosphorylation upon NRG-1 activation were verified by ELISA and WBs as described by our group previously [19 (link)].
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