EMT6 or 4T1 cells were harvested for protein lysates for immunoblotting as described in [31] (link). List of antibodies: anti-collagen I (ab34710, abcam), anti-beta-catenin (sc-7963, Santa Cruz Biotechnology), anti-alpha-tubulin (sc-8935, Santa Cruz Biotechnology), anti-phospho-LRP6 (#2568, Cell Signaling Technology), and anti-LRP6 (#3395, Cell Signaling Technology). Densitometry was determined by the ImageJ program.
Anti phospho lrp6
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
Anti-phospho-LRP6 is a primary antibody that specifically recognizes the phosphorylated form of the low-density lipoprotein receptor-related protein 6 (LRP6) cell surface receptor. LRP6 is a key component of the Wnt signaling pathway, which plays a critical role in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti lrp6, by Cell Signaling Technology (2 mentions)
Ab34710, by Abcam (1 mentions)
Sc 7963, by Santa Cruz Biotechnology (1 mentions)
Anti beta catenin, by Santa Cruz Biotechnology (1 mentions)
Anti alpha tubulin, by Santa Cruz Biotechnology (1 mentions)
Peroxide solution, by Merck Group (1 mentions)
Luminol, by Merck Group (1 mentions)
Anti phospho p65, by Cell Signaling Technology (1 mentions)
Anti phospho iκbα, by Cell Signaling Technology (1 mentions)
Anti wnt3a, by Cell Signaling Technology (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Anti phospho pten, by Cell Signaling Technology (1 mentions)
Ab83042, by Abcam (1 mentions)
Ab95230, by Abcam (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Ab8805, by Abcam (1 mentions)
Anti β catenin, by Cell Signaling Technology (1 mentions)
Anti phospho pi3k p85, by Cell Signaling Technology (1 mentions)
Anti pi3k p85, by Cell Signaling Technology (1 mentions)
Ab46154, by Abcam (1 mentions)
5 protocols using anti phospho lrp6
1
Protein Immunoblot Analysis of EMT6 and 4T1 Cells
2
Western Blot Analysis of Signaling Proteins
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The cells were lysed using RIPA lysate (1% NP40, 0.1% SDS, 5 mM EDTA, 0.5% sodium deoxycholate, and 1 mM sodium orthovanadate). Equal amounts (20 µg) of the samples were separated by SDS-PAGE and then transferred to a polyvinylidene fluoride membrane. The following primary antibodies (Cell Signaling Technology, Boston, MA, USA) were incubated with the membrane at 4 °C overnight: anti-ubiquitin-C (1:1000 dilution); anti-Wnt-3a (1:1000); anti-Wnt-7a (1:2000); anti-phospho-LRP6 (1:1000); anti-phospho-IκBα (1:1000); anti-phospho-p65 (1:1000); anti-phospho-PTEN (1:500); anti-phospho-Akt (1:1000) and anti-β-action (1:2000) antibodies. Secondary antibodies (Cell Signaling Technology, Boston, MA, USA, 1:5000 dilution) were added and incubated with enhanced chemiluminescence (ECL) reagent (including Luminol and Peroxide solution, Millipore, Schwalbach, Germany). The chemiluminescence, which reflected the expression of proteins, was visualized by X-ray film.
3
Corneal Protein Extraction and Western Blot
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Total protein from corneas was extracted on ice with RIPA lysis buffer in the presence of freshly added protease and phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA, USA). A total of 10 μg/lane protein extract was loaded onto a 4%–20% gradient SDS-polyacrylamide gel and transferred to nitrocellulose membranes (Bio-Rad Laboratories). Nonspecific binding was blocked with 5% nonfat milk or 5% BSA in Tris-buffered saline with Tween20 (TBST) as recommended for each antibody. The membrane was incubated with rabbit anti-VEGF (ab46154; Abcam, Cambridge, MA, USA), anti-Angpt1 (ab95230; Abcam), anti-Tie2 (catalog [Cat.] no. 7403; Cell Signaling, Danvers, MA, USA), anti-phospho-Tie2 (AF2720-SP; R&D Systems, Minneapolis, MN, USA), anti-PI3K (p85) (Cat. no. 4292; Cell Signaling), anti-phospho-PI3K (p85) (Cat. no. 4228; Cell Signaling), anti-Akt (ab8805; Abcam), anti-phospho-Akt1 (ab81283; Abcam), anti-Fzd4 (ab83042; Abcam), anti-LRP6 (Cat. no. 3395S; Cell Signaling), anti-phospho-LRP6 (Cat. no. 2568S; Cell Signaling), anti-N-p-β-catenin (Cat. no. 4270; Cell Signaling), or anti-β-catenin (Cat. no. 8480S; Cell Signaling) antibodies overnight at 4°C. IRDye 800CW goat anti-rabbit IgG (Cat. no. 926-32211; LI-COR, Lincoln, NE, USA) was used as the secondary antibody, and mouse anti-GAPDH antibody (ab8245; Abcam) was used as an internal standard.
4
Western Blot Analysis of Cell Signaling
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Lysates were prepared using RIPA buffer (10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 1% (v/v) Triton X-100, 0.1% (w/v) sodium deoxycholate, 0.1% (w/v) SDS and 140 mM NaCl) supplemented with protease inhibitor tablets (ThermoFisher, UK). Equal protein samples were separated on 10% (v/v) SDS polyacrylamide gel electrophoresis gels and transferred to PVDF membranes (Sigma Aldrich) which were blocked and probed overnight at 4 °C with 1:1000 anti-ERG (Abcam, UK), 1:10,000 anti-β-actin (Abcam), 1:2500 anti-GAPDH (Millipore, UK), 1:1000 β-catenin, 1:1000 anti-c-Myc, 1:1000 anti-cyclin D1 or 1:1000 anti-phospho-LRP6 (all Cell Signalling Technologies, UK) primary antibodies. Membranes were incubated in 1:2000 HRP-linked anti-rabbit or anti-mouse IgG secondary antibody (NEB) for 2 h at room temperature. Membranes were incubated in Luminata Forte Western HRP substrate (Millipore) for chemiluminescent detection prior to image acquisition using a Li-Cor Odyssey imaging system.
5
Comprehensive Panel of Breast Cancer Cell Lines
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MDA-MB-231, MCF7, T47D, SKBR3, BT474, BT-20, MDA-MB-468, Hs578t and MDA-MB-453 and MDA-MB-157 cells purchased from the American Type Culture Collection (Manassas, VA). MFM 223, SUM159PE and SUM185PE cells were kindly provided by Dr. Jennifer A. Pietenpol (Vanderbilt-Ingram Cancer Center). ACK1, AR, EGFR, Her2, Her3, Mcl-1, Bcl-2, Puma, YB-1, FAK, AKT, Anti-phospho-AKT (Ser473), anti-phospho-S6K1 (S79), Anti-phospho-4-EBP1, Anti-phospho-LRP6, Anti-phospho-mTOR, Anti-phospho-YB-1, Anti-phospho-FAK (Y397), anti- ribosomal protein S6, anti-ribosomal protein S6, BCL-xL and anti-cleaved Casp3 antibodies were obtained from Cell Signaling Technology (Beverly, MA). Anti-phospho-ribosomal protein S6 (S235/236), mouse anti-ERK antibody and Anti-phospho-ERK were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Vinculin was purchased from Sigma. Secondary anti-mouse IgG with horseradish peroxidase was from Calbiochem. Secondary anti-rabbit IgG with horseradish peroxidase was from GE Healthcare. Ceritinib and Paclitaxel were purchased from APExBIO. Bicalutamide (97%) purchased from ACROS Organics. The 133 FDA-approved drugs were kindly provided from NCI/DTP Approved Oncology Drugs Plated Set (AODVIII).
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