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)].
Anti p44 42 map kinase
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-p44/42 MAP kinase is a primary antibody product that binds to the p44/42 mitogen-activated protein (MAP) kinase proteins. MAP kinases are important signaling molecules that regulate cellular processes such as cell growth, proliferation, differentiation, and survival.
Automatically generated - may contain errors
Lab products found in correlation
Anti α tubulin, by Merck Group (2 mentions)
Ribonucleoside vanadyl complex, by New England Biolabs (1 mentions)
Bt474, by American Type Culture Collection (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti β tubulin, by Santa Cruz Biotechnology (1 mentions)
Hek293 freestyle cells, by Thermo Fisher Scientific (1 mentions)
Anti p akt, by Cell Signaling Technology (1 mentions)
Hek293, by American Type Culture Collection (1 mentions)
Hs587t, by American Type Culture Collection (1 mentions)
Rnase free dnase 1, by New England Biolabs (1 mentions)
Mda mb 468, by American Type Culture Collection (1 mentions)
Mcf 10a, by American Type Culture Collection (1 mentions)
Anti p erk1 2, by Cell Signaling Technology (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
Mda mb 231, by American Type Culture Collection (1 mentions)
Skbr3, by American Type Culture Collection (1 mentions)
Bt549, by American Type Culture Collection (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence, by Santa Cruz Biotechnology (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
4 protocols using anti p44 42 map kinase
1
HER3 Neutralizing Antibody Characterization
2
Protein Extraction and Western Blotting
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Protein extraction, analysis and western blotting were performed as previously described (20 (link)), applying the following primary antibodies: Polyclonal anti-CYLD [cat. no. 4495; dilution, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA; (21 (link))], anti-p44/42 MAP kinase (cat. no. 9102; dilution, 1:1,000) or anti-phospho-p44/42 MAP kinase (cat. no. 4370; dilution, 1:1,000; both Cell Signaling Technology, Inc.) and anti- β-actin (cat. no. A5441; dilution, 1:5,000; Sigma-Aldrich; Merck KGaA).
3
Quantitative Immunoblotting of Cellular Signaling
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Western blot analysis was performed as previously described (Shathasivam et al, 2015 (link)). Membranes were incubated with anti-VEPH1 (1 : 1000 or 1 : 2000, SDI Newark, DE, USA; or 1 : 250, Sigma), anti-Flag (1 : 500, Sigma), anti-AKT (1 : 1000, Cell Signaling Technology), anti-phospho(Ser473)-AKT (1 : 1000, Cell Signaling Technology), anti-p44/42 MAP Kinase (1 : 500, Cell Signaling Technology), anti-phospho(Thr202/Tyr204)-p44/42 MAP Kinase (1 : 500, Cell Signaling Technology), anti-GAPDH (1 : 1500, Santa Cruz Biotechnology, Dallas, TX, USA) or anti-α-tubulin (1 : 3000, Sigma) antibodies. Blots were visualised by enhanced chemiluminescence (Santa Cruz Biotechnology).
4
Western Blot Protein Expression Analysis
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Whole-protein extracts were analyzed using the Immuno Cruz Western blotting Luminol Reagent (Santa Cruz Biotechnology, Dallas, TX, USA) or the SuperSignal West Pico PLUS Chemiluminescent Substrate (ThermoFisher Scientific). The following antibodies were used: anti-GAPDH, anti-phospho-EGFR (Y1068), anti-phospho-AKT (S473), anti-phospho-p44/42 MAP kinase (T202/Y204), anti-AKT, anti-p44/42 MAP kinase, and anti-Snail (Cell Signaling Technology, Beverly, MA, USA); anti-EGFR, anti-E-cadherin, and anti-N-cadherin (Santa Cruz Biotechnology, Heidelberg, Germany); anti-vimentin antibody (DAKO, Milan, Italy); anti-α-tubulin (Sigma Aldrich, Milan, Italy). Densitometric analysis of the blots was performed using the ImageJ software v.1.8.0. Original blots were shown in Figures S5 and S6 .
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