Lysates were separated by SDS-PAGE and proteins transferred to PVDF membrane. Immunoreactivity was detected using horseradish protein (HRP) conjugate secondary antibodies (CalBioTech, Spring Valley, CA) and chemiluminescence substrate (ThermoScientific, Rockford, IL) on the Versadoc Imaging System (BioRAD, Hercules, CA). BRAF (sc-5284), ERK2 (sc-1647), and p-ERBB3 (Tyr1328, sc-135654) antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). ERBB3 (#4754), p-ERBB3 (Tyr1197, #4561), p-ERBB3 (Tyr1289, #4791), ERBB2 (#4290), p-ERBB2 (Tyr1196, #6942), p-ERBB2 (Tyr1221/1222, #2243), AKT (#9272), p-AKT (Ser473, #6942), p-AKT (Thr308, #2965), p-ERK1/2 (Thr202/Tyr204, #9101), and GAPDH (#2118) antibodies were purchased from Cell Signaling Technology (Beverley, MA). Actin (A2066) antibody was purchased from Sigma-Aldrich Co. (St. Louis, MO).
P erbb2
Manufactured by Cell Signaling Technology
Sourced in United States
P-ERBB2 is a phospho-specific antibody that detects the phosphorylated form of the HER2/ERBB2 protein. It is used for the detection and analysis of HER2/ERBB2 phosphorylation status in various experimental techniques.
Automatically generated - may contain errors
Lab products found in correlation
P akt, by Cell Signaling Technology (7 mentions)
Erbb2, by Cell Signaling Technology (6 mentions)
P erbb3, by Cell Signaling Technology (6 mentions)
P erk, by Cell Signaling Technology (5 mentions)
P erk1 2, by Cell Signaling Technology (4 mentions)
P egfr, by Cell Signaling Technology (4 mentions)
P stat3, by Cell Signaling Technology (4 mentions)
Erbb3, by Cell Signaling Technology (3 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
T erk, by Cell Signaling Technology (3 mentions)
P mtor, by Cell Signaling Technology (3 mentions)
Parp1, by Cell Signaling Technology (2 mentions)
P igf 1r, by Cell Signaling Technology (2 mentions)
Oct4a, by Cell Signaling Technology (2 mentions)
P p70s6k, by Cell Signaling Technology (2 mentions)
P70s6k, by Cell Signaling Technology (2 mentions)
β catenin, by Cell Signaling Technology (2 mentions)
4e bp1, by Cell Signaling Technology (2 mentions)
P 4ebp1, by Cell Signaling Technology (2 mentions)
Cyclin d1, by Santa Cruz Biotechnology (2 mentions)
15 protocols using p erbb2
1
Western Blot Analysis of Signaling Proteins
2
Maintenance of Breast Cancer Cell Lines
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MCF7 and anti-estrogen resistant derivatives (MCF7-T and MCF7-F) were maintained and established as we have previously described [30 (link)]. Briefly, MCF7-T cells were maintained in tamoxifen-containing media, except prior to drug treatment. The maintenance concentration of tamoxifen was 100 nM. MCF7/LCC2 and MCF7/LCC9 cells lines (anti-estrogen resistant) were derived and maintained as described by Brünner et al. [33 (link)]. Cell lines were authenticated in 2017 by ATCC and tested for mycoplasma contamination (Manassas, VA, USA). To ensure cell line integrity, all cell lines were thawed at frequent intervals and not used beyond 30 passages. Additionally, cell morphology was monitored for each cell line, and proper media and growth conditions selected [29 (link),30 (link)]. Hydrogen peroxide (H2O2) was purchased from EMD Millipore (Billerica, MA, USA). Tamoxifen (Tamox or 4-OHT) was purchased from Sigma Aldrich (St. Louis, MO, USA). Talazoparib (Talaz; PARPi) was provided by Pfizer/Medivation (San Francisco, CA, USA). Primary antibody dilutions were as followed: PARP1 (Cell Signaling, Danvers, MA, USA; 1:3000), PAR (Trevigen, Gaithersburg, MD, USA; 1:2000) ERα (Santa Cruz, Dallas, TX, USA; 1:2000), pERBB2 (Cell Signaling; 1:2000), GAPDH (Santa Cruz; 1:2000), IgG (Santa Cruz; 1:5000), β-Tubulin (Santa Cruz; 1:5000).
3
Protein Expression Analysis by Western Blot
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Whole-cell lysates were prepared using EBC lysis buffer (50-mM Tris–HCl [pH 8.0], 120-mM NaCl, 1% Triton X-100, 1-mM EDTA, 1-mM EGTA, 0.3-mM phenylmethylsulfonylfluoride, 0.2-mM sodium orthovanadate, 0.5% NP-40, and 5-U/mL aprotinin) and were then centrifuged. Proteins were separated using SDS-PAGE and transferred to PVDF membranes (Invitrogen) for Western blot analysis. Membranes were probed using antibodies against p-ALK (Tyr1604), ALK, p-Akt (Ser473), p-MET (Tyr1234/1235), p-ErbB2 (Tyr1221/1222), ErbB2, p-ErbB3 (Tyr1289), ErbB3, p-IGF1R (Tyr1135/1136), p-Erk (Thr202/Tyr204), caspase-3, PARP-1 (all from Cell Signaling Technology, Beverly, MA) Akt, Erk, p-EGFR (Tyr1173), EGFR, MET, and β-actin (all from Santa Cruz Biotechnology, Santa Cruz, CA) as the first antibody; the membranes were then treated with a horseradish peroxidase-conjugated secondary antibody. All membranes were developed using an enhanced chemiluminescence system (Thermo Scientific, Rockford, IL). Densitometric analysis was performed using the ImageJ software provided by NIH (http://rsb.info.nih.gov/nih-image/ ).
4
Protein Expression Analysis in Heart Tissues
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Total proteins isolated from the heart tissues were size fractionated using SDS-PAGE and transferred onto Immobilon-P membranes (Millipore, Billerica, MA). The blotted membranes were incubated with antibodies against p-ERK, t-ERK, p-ErbB2, p-ErbB4, LC3b (Cell Signaling Technology, Beverly, MA), β1-AR (beta-1 adrenergic receptor; Abcam, Cambridge, MA), C/EBPβ, and AT1-R, (Santa Cruz Biotechnology Inc, Santa Cruz, CA) and with an HRP-conjugated secondary antibody (1:5000, KangChen Biotechnology, Shanghai, China). Either GAPDH or t-ERK was used as an internal control. The proteins were visualized using an ECL Western blotting detection system (GE Healthcare, catalog number RPN2106). The relative intensities of the protein bands were analyzed through densitometry with a gel documentation system using LAS-300 image analysis software. All experiments were repeated at least 3 times.
5
Western Blot Analysis of Protein Signaling
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Cells were harvested and lysed in NETN (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% NP40) for 20 min on ice. Lysates were cleared by centrifugation at 13 200 r.p.m. at 4 °C for 5 min. Supernatants were collected and protein concentrations were determined by the Bradford assay (Bio-Rad, Hercules, CA, USA). The proteins were then separated with a SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (Bio-Rad). After blocking in TBS with 5% nonfat milk for 1 h, the membranes were incubated overnight at 4 °C with the primary antibodies in TBST with 1% BSA. The following antibodies were purchased from Cell Signaling (Beverly, MA, USA): p-ERK1/2, t-ERK, p-ErbB2, and tubulin. Antibodies were purchased from other companies as follows: β-actin antibody (Sigma), MST4 (Abcam, Cambridge, MA, USA), t-ErbB2 (Calbiochem, Billerica, MA, USA). Membranes were extensively washed with TBST and incubated with horseradish peroxidase conjugated secondary anti-mouse antibody or anti-rabbit antibody (dilution 1 : 2000, Bio-Rad). After additional washes with TBST, antigen–antibody complexes were visualized with the enhanced chemiluminescence kit (Pierce, Rockford, IL, USA).
6
AZD4547 Receptor Signaling Profiling
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AZD4547 was purchased from LC Laboratories (Woburn, MA). FGFR1, FGFR2, p-FGFR, EGFR, p-EGFR, ErbB2, p-ErbB2, Akt, p-Akt, p-Erk1/2, mTOR, p-mTOR, 4EBP1, p-4EBP1, p70S6K, p-p70S6K, β-catenin, p-β-catenin, Dvl2, Oct4A, and p-Lrp6 primary antibodies were purchased from Cell Signaling (Danvers, MA). Erk2, p27, and β-actin primary antibodies, BGJ398, and SU5402 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Primary antibodies used for IF staining, including Cytokeratin 14 (K14), Cytokeratin 8/18 (K8/18), and α-smooth muscle actin (SMA) were purchased from Leica Biosystems (Buffalo Grove, IL), Developmental Studies Hybridoma Bank (DSHB; University of Iowa, Iowa City, IA), and Sigma (St. Louis, MO), respectively.
7
Buformin Effects on Cell Signaling
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Buformin was purchased from Wako Pure Chemical Industries (Osaka, Japan). Primary antibodies against AMPK, p-AMPK, mTOR, p-mTOR, p70S6K, p-p70S6K, 4EBP1, p-4EBP1, IRS, p-IRS, IGF1R, p-IGF1R, p-erbB-2, Akt, p-Akt, p-Erk1/2, p-Stat3, p-ER, β-catenin, Oct4A, and Notch were purchased from Cell Signaling (Danvers, MA). Antibodies against IGF1Rα/β, Erk, Stat3, ER, Cyclin D1, and β-actin were ordered from Santa Cruz Biotechnology (Santa Cruz, CA). erbB-2 and active β-catenin antibodies were purchased from EMD Millipore (Billerica, CA).
8
Characterization of Ovarian Cancer Cell Lines
9
Western Blot Analysis of EMT-related Proteins
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Total proteins from cultured cells were lysed using RIPA buffer supplemented with protease and protease inhibitors (Roche, Mannheim, Germany). An equal amount of 50 μg proteins were fully electrophoresed on 6–10% SDS polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The membranes were incubated with primary antibodies at 4 °C overnight after blocked with 5% non-fat milk at room temperature for 1 h. The primary antibodies against GAPDH were obtained from Abcam (Cambridge, MA, USA). The antibodies against E-cadherin, N-cadherin, Vimentin, Slug, Zeb1, NRG1, ERBB2, p-ERBB2, ERBB3, p-ERBB3, MEK1, p-MEK1, Erk, p-Erk, Raf, p-Raf, Fra-1, Fra-2, c-Fos, FosB, c-Jun and JunB (diluted at 1:1000 ratio) were purchased from Cell Signaling Technology (Beverly, MA, USA). Then, the membranes were incubated with goat anti-rabbit or anti-mouse secondary antibody (diluted at 1:5000 ratio) and then detected with ECL Detection System (Thermo Scientific, Rockford, IL, USA).
10
Ganetespib-Induced Apoptosis Pathway
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Ganetespib was purchased from Medkoo Biosciences, Inc. (Chapel Hill, NC, USA). Primary antibodies specific to Cyclin B1, cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, cleaved PARP, Akt, phospho Akt (pAkt), mTOR, pmTOR, ErbB2, pErbB2, GSK3, pGSK3, Erk, pErk, Src and pSrc were purchased from Cell Signaling Technology (Danvers, MA, USA); and cyclin D1, cyclin E, Cdk1, E2F1, p27, survivin, caspase-8, caspase-9, EGFR and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary anti-mouse or anti-rabbit antibodies were purchased from Thermo Scientific (Rockford, IL, USA).
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