The PAPP-A-mediated cleavage of endogenous IGFBP-4 in ascites was further assessed by incubating ascites samples (n = 10) with 0.1 nM recombinant PAPP-A [19 (link)] or buffer at 37°C for 6 h. Ascites samples containing the highest amounts of intact IGFBP-4 were chosen. Buffer containing rhIGF-I (0 or 5 ug/l) or rhIGF-I (5 ug/l) and PAPP-A (0.1 nM) were incubated and served as controls. The reaction mixtures were immediately used for measurements of bioactive IGF using the KIRA bioassay. In addition, IGFBP-4, C-terminal and N-terminal IGFBP-4 were determined in the reaction mixtures using immunoassays as previously described [22 (link), 23 (link)]. Finally, to assess the intracellular signalling pathways initiated by IGF-IR activation, cell lysates from the IGF bioactivity measurements were separated by 4–15% SDS-PAGE and transferred to PVDF membranes. Levels of phosphorylation of the intracellular proteins Akt, mTOR and S6 were determined by probing the blots with anti-p-Akt antibody (AF887, R&D Systems, Abingdon, UK), anti-p-TOR antibody (AF1665, R&D Systems) and anti-p-S6 antibody (AF3918, R&D Systems). Total IGF-IR levels in the cell lysates were determined using anti-hIGF-IR antibody (MAB391, R&D Systems) and used as loading controls. Additionally, stain-free total protein quantitation using the ChemiDoc™ system (Bio-Rad) served as total protein loading control.
Mab391
Manufactured by R&D Systems
Sourced in United States
MAB391 is a mouse monoclonal antibody that recognizes human PCSK9. PCSK9 is a serine protease that plays a role in regulating low-density lipoprotein (LDL) receptor levels.
Automatically generated - may contain errors
Lab products found in correlation
Ldk378, by Selleck Chemicals (2 mentions)
Crizotinib, by Chemietek (2 mentions)
Lapatinib, by Selleck Chemicals (2 mentions)
Aew541, by Selleck Chemicals (2 mentions)
Osi 906, by Selleck Chemicals (2 mentions)
Chemidoc system, by Bio-Rad (1 mentions)
Af887, by R&D Systems (1 mentions)
Dulbecco s modified eagle s medium f12 dmem f12, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Merck Group (1 mentions)
Lightcycler 480 sybr green 1 master, by Roche (1 mentions)
Insulin, by Merck Group (1 mentions)
Igf 1, by Merck Group (1 mentions)
Its premix, by BD (1 mentions)
Pd123319, by Merck Group (1 mentions)
Candesartan, by AstraZeneca (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Irdye 680 conjugated goat polyclonal anti rabbit igg, by LI COR (1 mentions)
Irdye 800cw conjugated goat polyclonal anti mouse igg, by LI COR (1 mentions)
Ab22552, by Abcam (1 mentions)
Ba 9500, by Vector Laboratories (1 mentions)
6 protocols using mab391
1
PAPP-A-Mediated IGFBP-4 Cleavage and IGF Signaling
2
Preparation and Dissolution of Kinase Inhibitors
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X-376 was prepared as described previously14 (link). Crizotinib (ChemieTek, Indianapolis, IN, USA), OSI-906 (Selleck Chemicals, Houston, TX, USA), AEW-541 (Selleck Chemicals, Houston, TX, USA), LDK-378 (Selleck Chemicals, Houston, TX, USA) and Lapatinib (Selleck Chemicals, Houston, TX, USA) were dissolved in DMSO. Erlotinib was synthesized by the Memorial Sloan-Kettering Cancer Center (MSKCC) Organic Synthesis Core. MAb391 (R&D systems, Minneapolis, MN, USA) was dissolved in PBS.
3
Preparation and Dissolution of Kinase Inhibitors
4
Investigating Ang II-Mediated Signaling Pathways
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Human recombinant Ang II, insulin, IGF-1, and AT2 receptor blocker PD123319 were procured from Sigma-Aldrich (St. Louis, MO, USA). PKC inhibitor Gö6983, PI3K inhibitor LY294202, and MEK1/2 inhibitor U0126 were purchased from Calbiochem (San Diego, CA, USA). AT1 receptor blocker candesartan was from AstraZeneca. Neutralizing anti-IGFR1 antibody, MAB 391, was bought from R & D Systems (Minneapolis, MN, USA). Dulbecco's modified Eagle's medium/F12 (DMEM/F12) was from Life Technologies (Carlsbad, CA, USA). Nu serumTM and ITS+premix were obtained from BD Biosciences (Bedford, MA, USA). Anti-phospho-ERK1/2 (Thr202/Tyr204), anti-phospho-AKT (Ser473), anti-ERK1/2, and anti-AKT antibodies were from Cell Signaling Technology (Danvers, MA, USA). Secondary antibodies [IRDye 800CW Conjugated Goat (polyclonal) anti-mouse IgG and IRDye 680 Conjugated Goat (polyclonal) anti-rabbit IgG] were products of LI-COR Biosciences (Lincoln, NE, USA). High-capacity cDNA reverse transcription kit was from Applied Biosystems. LightCycler® 480 SYBR Green I Master was purchased from Roche Applied Science. Trizol reagent and all other reagents came from Sigma-Aldrich (St. Louis, MO, USA).
5
Immunohistochemical Analysis of DT Receptor and Osteoblast Markers
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Expression of the DT receptor was detected by immunohistochemistry using an anti-human heparin-binding EGF-like growth factor (anti-hHB-EGF) antibody. In brief, paraffin sections were treated with 0.1% trypsin and 3% H2O2, and blocked with reagent containing 5% animal serum in PBS + Tween 20 for 1 hr. Anti-hHB-EGF antibody (R&D Systems MAB391, 1:25 dilution) was used as primary antibody coupled with biotinylated IgG secondary antibody (Vector Laboratories BA-9500, 1:400 dilution). After staining, sections were treated with a Vectastain ABC Kit for biotin-streptavidin signal amplification and visualized by a DAB Peroxidase Substrate Kit. Osx+ cells were detected by staining sections with anti-SP7 antibody (Abcam Ab22552, 1:2,000 dilution) coupled with secondary antibody conjugated to Alexa Fluor 488 (Thermo Fisher Scientific A27034, 1:1,000 dilution). Ocn+ cells were recognized by anti-OCN antibody (Santa Cruz Biotechnology SC18322, 1:50 dilution) followed by secondary antibody conjugated to Alexa Fluor 488 (Thermo Fisher A11078, 1:1,000 dilution). Green fluorescent OSX- or OCN-antibody staining was then individually superimposed with red fluorescent TUNEL staining (In Situ Cell Death Detection Kit TMR Red, Roche Applied Science).
6
Automated Quantification of IGF-1R Inhibition
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MCF-7 cells were seeded in 96-well optical plate and maintained in normal DMEM or phenol-red free DMEM. Cells were treated for 3 days with 11 µg/mL IGF-1R antibody (R&D Systems, MAB391). After that, cells were fixed with 4% formaldehyde in PBS 1× for 30 minutes at room temperature. Cells were washed with PBS (3×5) then incubated with permeabilization solution (0.5% Triton X-100 in PBS) for 40 minutes at room temperature, followed by washing steps. Cell nuclei were stained with DAPI (1 µg/mL) for 15 minutes at room temperature. All cells were counted by automated fluorescent microscopy (LEICA DMi8) using the LasX software based on DAPI signal, representing cell nuclei. Image analysis was performed using ImageJ software.
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