Lipofectamine 2000® and TRIzol® were obtained from Invitrogen (Carlsbad, CA). Polybrene was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-HA antibodies, 17β-estradiol, 4-hydroxytamoxifen, and puromycin were purchased from Sigma (St. Louis, MO). Anti-actin was purchased from BD Bioscience (San Jose, CA). The ErbB2/HER2, ERα, and pSer118 ERα antibodies were purchased from Cell Signaling (Danvers, MA). LDE-225, LY2940002, and lapatinib ditosylate were purchased from Selleckchem (Houston, TX). Rapamycin was obtained from Fisher Scientific (Waltham, MA). Blasticidin S Hydrochloride was obtained from MP Biomedicals (Santa Ana, CA). 0.4% Trypan Blue Solution was purchased from Cellgro (Manassas, VA). Recombinant human Shh(C24II) was purchased from R&D Systems (Minneapolis, MN).
Her2 erbb2
Manufactured by Cell Signaling Technology
Sourced in United States, Germany
HER2/ErbB2 is a cell surface receptor tyrosine kinase that is part of the ErbB family of receptors. It plays a key role in the regulation of cell growth and survival.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Her3 erbb3, by Cell Signaling Technology (2 mentions)
Total akt, by Cell Signaling Technology (2 mentions)
Pegfr y1068, by Cell Signaling Technology (2 mentions)
Total egfr, by Cell Signaling Technology (2 mentions)
Lapatinib ditosylate, by Selleck Chemicals (1 mentions)
Rapamycin, by Thermo Fisher Scientific (1 mentions)
Polybrene, by Santa Cruz Biotechnology (1 mentions)
Blasticidin s hydrochloride, by MP Biomedicals (1 mentions)
Trypan blue solution, by Corning (1 mentions)
Recombinant human shh c24ii, by R&D Systems (1 mentions)
Anti ha antibody, by Merck Group (1 mentions)
Lde225, by Selleck Chemicals (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
17β estradiol, by Merck Group (1 mentions)
4 hydroxytamoxifen, by Merck Group (1 mentions)
Puromycin, by Merck Group (1 mentions)
Anti actin, by BD (1 mentions)
Nuclear and cytoplasmic protein extraction kit, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
9 protocols using her2 erbb2
1
Reagents for Cell Signaling Studies
2
Protein Expression Analysis in Myocardial Tissue
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Cytoplasmic and nuclear protein were extracted using the Nuclear and Cytoplasmic Protein Extraction Kit (Thermo Scientific). Myocardial protein extracts were resolved by SDS/PAGE and transferred onto a polyvinylidene difluoride membrane (Millipore). Membranes were blocked with 5% milk in Tris-buffered saline with Tween 20 for 2 h at 37 °C and then incubated overnight at 4 °C with primary antibodies. The primary antibodies used for all Western blots were ErbB2/HER2 (Cell signaling), phosphatidylinositol 3 kinase (PI3K, Cell signaling), phospho-PI3K (Cell signaling), protein kinase B (Akt, Cell signaling), phospho-Akt (Ser473) (Cell signaling), phospho-IκBα (Cell signaling), nuclear factor kappa B (NF-κB, Cell signaling), iNOS (Abcam), histone (Cell signaling), or β-actin (GTX-109639, GeneTex).
3
Cryopreserved Cell Immunophenotyping Protocol
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The cryopreserved suspension was rapidly thawed. Cells were first filtered with a 70 µm filter, blocked with blocking buffer, and then stained with the appropriate conjugated antibodies for 30 min in the dark at 4° C. In some cases, cells were fixed using 10% formalin at 37° C prior to antibody staining. In unfixed cases, cells were counterstained with DAPI for viability. Antibodies were washed off with phosphate-buffered saline prior to flow cytometric analysis. Antibodies used were EpCAM/CD326 (BioLegend #324208, 1:20 dilution), CD45 (Biolegend #304034, 1:20 dilution), and HER2/ErbB2 (Cell Signaling #98710, 1:20 dilution). Gating strategies for the flow cytometric data are shown in Supplementary Fig. 2 .
4
Antibody and Protein Assay Protocol
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Anti-MSI2 (#ab76148), anti-MSI1 (#ab21628), and anti-β-actin HRP conjugated (#ab49900) antibodies and recombinant MSI2 protein (#ab167853) were obtained from Abcam, (Cambridge, UK). Anti-EGF receptor (#4267), phospho-EGFR (Y1068) (mAb #2234), HER3/ErbB3 (#12708), HER2/ErbB2 (#4290), Smad3 (#9523), phospho-AKT (T308) (#13038), total AKT (#2920), phospho-ERK (T202/Y204) (#4370), total ERK (#4696), phospho-p70S6K (T389) (#9234), total p70S6K (#2708), and normal Rabbit IgG (#2729) were obtained from Cell Signaling, (Danvers, MA). Erlotinib and afatinib were obtained from LC Laboratories (Woburn, MA), doxycycline from Sigma-Aldrich (D9891, Darmstadt, Germany). SUPERase-In RNAse inhibitor was obtained from Thermo Fisher Scientific, (AM2694 Waltham, MA). Osimertinib was obtained from Selleckchem (#S7297, Houston, TX)
5
Comprehensive Immunoblot Analysis
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Immunoblot analysis was performed as previously described (7 (link)). Primary Abs against p-ERK1/2 T202/Y204 (catalog 4370), total ERK1/2 (catalog 4695), p-AKT S473 (catalog 4060), total AKT (catalog 9272), p-Rb S807/811 (catalog 8516), total Rb (catalog 9309), p-p90RSK T359/S363 (catalog 9344), RSK1 (catalog 8408), p-HER2/ErbB2 Y1221/1222 (catalog 2243), HER2/ErbB2 (catalog 2165), p-EGFR Y1068 (catalog 3777), cyclin D1 (catalog 2922), CDK4 (catalog 12790), CDK6 (catalog 13331), vinculin (catalog 18799), and β-actin (catalog 12620) were purchased from Cell Signaling Technology. Pierce HRP-conjugated secondary Abs (anti-rabbit and anti-mouse) were purchased from Thermo Fisher Scientific. Amersham ECL Prime chemiluminescent detection reagent (GE Healthcare Life Sciences) was used to visualize protein expression.
6
Quantifying Molecular Markers in Cancer Cells
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IGFBP3, SPHK1 and CD44 mRNA expression was measured, in duplicate, on duplicate RNA extracts by qRT-PCR as previously described [13 (link)], using the following Taqman probes (Applied Biosystems, Foster City, CA, USA): IGFBP-3: Hs00181211_m1; SPHK1: Hs00184211_m1; CD44: Hs01075864_m1; and HMBS (reference gene): Hs00609297_m1. IGFBP-3 concentrations in cell-conditioned media were measured by in-house radioimmunoassay [13 (link)]. Western blotting was performed as described previously [13 (link)] using antibodies from Cell Signaling Technology (Beverly, MA, USA): total EGFR (#2232, 1:1,500), pEGFR (Tyr1068) (#2234, 1:1,500), HER2/ErbB2 (#2242, 1:1,000), type 1 IGF receptor (IGF1R, #3027, 1:1,000), p53 (#9282, 1:1,000), p63 (#4892, 1:1,000), CD44 (#3570, 1:1,500), vimentin (#3390, 1:1,000), E-cadherin (#3195, 1:1,000). SphK1 antibody (ab16491, 1:1,000) was from Abcam (Walnut, CA, USA) and α-tubulin antibody (T9026, 1:10,000) from Sigma-Aldrich, St Louis, MO, USA. Rabbit antihuman IGFBP-3 antiserum R-100 was raised in-house. Secondary antibodies were from Pierce Biotechnology (Rockford, IL, USA).
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IGFBP3, SPHK1 and CD44 mRNA expression was measured, in duplicate, on duplicate RNA extracts by qRT-PCR as previously described [13 (link)], using the following Taqman probes (Applied Biosystems, Foster City, CA, USA): IGFBP-3: Hs00181211_m1; SPHK1: Hs00184211_m1; CD44: Hs01075864_m1; and HMBS (reference gene): Hs00609297_m1. IGFBP-3 concentrations in cell-conditioned media were measured by in-house radioimmunoassay [13 (link)]. Western blotting was performed as described previously [13 (link)] using antibodies from Cell Signaling Technology (Beverly, MA, USA): total EGFR (#2232, 1:1,500), pEGFR (Tyr1068) (#2234, 1:1,500), HER2/ErbB2 (#2242, 1:1,000), type 1 IGF receptor (IGF1R, #3027, 1:1,000), p53 (#9282, 1:1,000), p63 (#4892, 1:1,000), CD44 (#3570, 1:1,500), vimentin (#3390, 1:1,000), E-cadherin (#3195, 1:1,000). SphK1 antibody (ab16491, 1:1,000) was from Abcam (Walnut, CA, USA) and α-tubulin antibody (T9026, 1:10,000) from Sigma-Aldrich, St Louis, MO, USA. Rabbit antihuman IGFBP-3 antiserum R-100 was raised in-house. Secondary antibodies were from Pierce Biotechnology (Rockford, IL, USA).
7
Signaling Pathway Protein Analysis
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Antibodies to pAKT (cat# 4060), total AKT (cat# 9272), pEGFR(Y845) (cat #2231), pEGFR(Y1092), pEGFR (Y1068) (cat# 3777), pEGFR (Y1173), total EGFR (cat # 4267; # 2232), Her2/ErbB2, c-Met, pFAK, FAK, pErk1/2 (42/44) (cat # 9101; cat # 4370), Erk1/2 (42/44) (cat # 4695) and GAPDH (all from Cell Signaling), β-adaptin (BD Biosciences, cat # 610382), EEA1 (Ab70521), and Actin were from Abcam. total EGFR antibody (Sc-03) was from SantaCruz Biotechnology. The HRP-conjugated secondary antibodies were from Jackson ImmunoResearch. Recombinant human EGF (AF-100-15) was obtained from PeproTech, custom BUB1 siRNA, as well as non-silencing siRNA (NSS) were obtained from GE-Dharmacon. SD208 (Tocris), Erlotinib (gift from Genentech), Cetuximab (Bristol Myer Squibb), 2OH-BNPP1 was synthesized in-house as described by Jiang et al., [40 ].
Cycloheximide was obtained from Acros Organics/Fischer Scientific, while DSS was from ThermoFisher Scientific. FuGENE 6 was from Roche while Lipofectamine 2000 was from Invitrogen/ThermoFisher Scientific. ProLong Gold anti-fade mounting media (without DAPI) was purchased from Invitrogen/ThermoFisher Scientific.
Cycloheximide was obtained from Acros Organics/Fischer Scientific, while DSS was from ThermoFisher Scientific. FuGENE 6 was from Roche while Lipofectamine 2000 was from Invitrogen/ThermoFisher Scientific. ProLong Gold anti-fade mounting media (without DAPI) was purchased from Invitrogen/ThermoFisher Scientific.
8
Immunoblotting Analysis of Cellular Proteins
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Cells were lysed in RIPA buffer. Equal amounts of total protein were resolved by SDS-PAGE, transferred to PVDF membranes (Millipore), and immunoblotted with the indicated primary antibodies. Membranes were hybridized with the following primary antibodies: p-Erk1/2, Erk1/2, p-HER3/ErbB3, HER3/ErbB3, HER2/ErbB2, p-AKT, AKT (Cell Signaling Technology), NIS, Tg, TPO, TSHR, GLUT1, and GLUT3 (Protein tech). Membranes were then hybridized with species-specific HRP-conjugated antibodies. Bands were visualized with Potent ECL kit (Beyotime).
9
Immunoblotting Analysis of Cell Signaling
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All used primary antibodies for Chk1, phospho-Chk1 S345, Estrogen Receptor-α, phospho-Estrogen Receptor-α S118, HER2/ErbB2 and phospho-HER2/ErbB2 Y1248 were purchased from Cell Signaling (Frankfurt, Germany). Secondary antibodies anti-mouse IgG, HRP conjugated and anti-rabbit IgG, HRP conjugated, were purchased from Pierce (Bonn, Germany).
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