λ-Phosphatase treatment,19 (link) GST pull down,19 (link) far western,19 (link) protein extracts preparation,12 (link) immunoprecipitation,12 (link) immunoblotting assays12 (link) and biotinylation assay12 (link), 62 (link) were performed as previously described. Immunoblot analysis was performed using the following antibodies: anti-Flag (Sigma, Cat#F3165), anti-Flag-HRP (Sigma; Cat#A8592), anti-MPM2 (05-368, Millipore), anti-Notch1Val1744 (Cell Signaling, Danvers, MA, USA, Cat#2421), anti-Notch3 (Cell Signaling; Cat#2889); anti-Notch3 M20 (Santa Cruz Biotechnology, Dallas, TX, USA, Cat# sc-7424), anti-Pin1 (Santa Cruz Biotechnology; Cat#sc-46660), anti-β-actin (Santa Cruz Biotechnology; Cat#sc-47778), anti-Lck (Santa Cruz Biotechnology; Cat#sc-433), anti-α-tubulin (Santa Cruz Biotechnology; Cat#sc-803), anti-LaminB M20 (Santa Cruz Biotechnology; Cat#sc-6217) and anti-Hes1 (Santa Cruz Biotechnology; Cat#sc-25392). The antibody against the activated Notch3-IC protein (N3IC-act) was kindly provided by Genentech (South San Francisco, CA, USA). The anti-N3EC (5E1) antibody was kindly provided by Professor A Joutel.62 (link) The anti-pTα antibody was kindly provided by H von Bohemer.63 (link)
Anti mpm2
Manufactured by Merck Group
Sourced in United States
Anti-MPM2 is a laboratory equipment product developed by Merck Group. It is designed to detect and quantify the presence of the mitotic phosphoprotein monoclonal antibody 2 (MPM-2) in biological samples. The core function of Anti-MPM2 is to serve as a tool for researchers and scientists to study cell cycle progression and cell division processes.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Anti flag, by Merck Group (2 mentions)
Facscanto 2, by BD (2 mentions)
Facsdiva software, by BD (2 mentions)
Facscalibur, by BD (2 mentions)
Anti brdu, by Abcam (2 mentions)
Benzonase nuclease, by Merck Group (2 mentions)
Clarity ecl, by Bio-Rad (2 mentions)
Anti mouse igg hrp, by Cell Signaling Technology (2 mentions)
Nupage 4 12 bis tris protein gel, by Thermo Fisher Scientific (2 mentions)
Anti rabbit igg hrp, by Cell Signaling Technology (2 mentions)
Ab21686, by Abcam (2 mentions)
B 5 1 2, by Merck Group (2 mentions)
Anti h3s10ph, by Merck Group (2 mentions)
Anti vrk2, by Santa Cruz Biotechnology (2 mentions)
Anti vinculin, by Cell Signaling Technology (2 mentions)
Anti α tubulin, by Santa Cruz Biotechnology (1 mentions)
Anti notch3 m20, by Santa Cruz Biotechnology (1 mentions)
Anti flag hrp, by Merck Group (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
11 protocols using anti mpm2
1
Protein Interaction Analysis Techniques
2
Cell Cycle Analysis by Flow Cytometry
3
Cell Cycle Analysis by Flow Cytometry
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Cells were harvested and fixed in 70% ethanol for at least 2 h at -20ºC before immunostaining and flow cytometry. Combined analysis of DNA content with propidium iodide (PI), BrdU (anti-BrdU, Abcam, Cambridge, United Kingdom, ab6326; 1/250) positive population and the MPM2 (anti-MPM2, Millipore, Burlington, MA, USA, #05–368; 1/250) positive population were performed with a BD FACSCalibur (Cytometry and Cell Sorting Core Facility, IDIBAPS) as previously described [50 (link)].
4
Western Blotting Analysis of Cell Signaling
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Western blotting assay was performed as described previously (Cao et al, 2011; Zhou et al, 2009). The following antibodies were used in this study: anti-FOXM1 (Santa Cruz, sc-500); anti-acetylated lysine (Cell Signaling Technology, #9681); anti-CDK1 (Abcam, ab133327); anti-MPM-2 (Millipore, 05-368); anti-p300 (Santa Cruz, sc-584); anti-Plk1 (Santa Cruz, sc-17783); anti-CDC25B (Santa Cruz, sc-326); anti-CyclinB1 (Santa Cruz, sc-245); anti-Aurora B (Cell Signaling Technology, 3049S); anti-FLAG (Sigma); anti-Survivin (Cell Signaling Technology, 2808); anti-HA-tag (Cell Signaling Technology); anti-GAPDH (Santa Cruz, sc-47724).
5
Cell Cycle Analysis by Flow Cytometry
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Cells were fixed in 70% ethanol overnight at 4°C and resuspended in propidium iodide (5 μg/ml) and RNaseA (1 μg/ml) in PBS. FACS analyses were performed on a BD FACSCanto II (BD Biosciences), and 10,000 events were counted. Data analyses were performed using the BD FACSDiva software (BD Biosciences) and adapted with FlowJoTM 10.7.1. Representative examples of cell cycle profiles that are shown in the figures were repeated at least three times. The mitotic index was determined by staining of fixed cells with anti-MPM2 (1:1,600; Millipore) and secondary antibodies conjugated to Alexa Fluor 488 (1:2,000; Molecular Probes) as described previously (91 (link)).
6
Western Blot Analysis of Mitotic Regulators
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Cells were washed once in PBS and then lysed in 141 mM Tris Base, 106 mM Tris HCl, 2% LDS, 1% Benzonase nuclease (E1014, Millipore), pH 8.5, at room temperature for 15 min and snap frozen in liquid nitrogen. The protein concentration was determined using Rapid Gold BCA Protein Assay Kit (Pierce). Cell lysates (20 µg) were run on NuPAGE 4–12% Bis–Tris protein gels (Invitrogen) using standard procedures, transferred to 0.2 µm polyvinylidene fluoride membranes and blocked in 5% milk in TBST (0.05% Tween 20 in Tris-buffered saline) for 1 h at room temperature before overnight incubation with primary antibodies. Membranes were washed thrice in TBST then incubated with a HRP conjugated secondary antibody for 1 h at room temperature in TBST with 5% milk, and signals were detected by chemiluminescence (Clarity ECL, Biorad). Antibodies used in immunoblotting were as follows: rabbit polyclonal anti-Vinculin (Cell Signaling Technology, #13901), anti-Haspin (Abcam, ab21686, lot 156753), and anti-H3T3ph (B86349 (link)) with anti-rabbit IgG HRP (Cell Signaling Technology, #7074) and mouse monoclonal anti-VRK1 (Abcam, ab171933), anti-VRK2 (Santa Cruz, sc-365199), anti-H3S10ph (Millipore, 05-806), anti-α-tubulin (B-5-1-2, Sigma, T5168) and anti-MPM2 (Millipore, 05-368) with anti-mouse IgG HRP (Cell Signaling Technology, #7076).
7
PBMC Activation and Mitotic Arrest Protocol
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PBMCs were extracted from blood samples by centrifugation on a Ficoll cushion (Cytiva) for 30 min at 1800 rpm at room temperature (RT) and washed several times in phosPHAte-buffered saline (PBS). After 30 min of adhesion step at 37°C in RPMI 1640, nonadherent cells were cultured in RPMI 1640 in the presence of PHA (5 μg/ml; Sigma-Aldrich) to induce T lymphocyte proliferation. After 2 days, PHA was washed, and IL-2 (50 U/ml; STEMCELL Technologies) was added to the medium and replaced every 2 days for a time period of 5 days.
To study mitotic progression, PBMCs were synchronized in G2 by overnight incubation with 5 μM RO-3306 (Selleck Chemicals, S7747). Cell cycle arrest was released by washing cells twice in PBS, and 1 μM taxol (Selleck Chemicals, S1150) was subsequently added to arrest cells in mitosis. Flow cytometry analysis was performed on PBMCs fixed with 4% paraformaldehyde (PFA), permeabilized with 0.5% Triton X-100–PBS, and stained with anti-MPM2 (Millipore, 05-368). Cells were washed with PBS and incubated with a secondary antibody conjugated to Alexa Fluor 488 (Molecular Probes). Secondary antibody was washed with PBS, and DNA was stained with DAPI (2 mg/ml) for 30 min at 37°C.
To study mitotic progression, PBMCs were synchronized in G2 by overnight incubation with 5 μM RO-3306 (Selleck Chemicals, S7747). Cell cycle arrest was released by washing cells twice in PBS, and 1 μM taxol (Selleck Chemicals, S1150) was subsequently added to arrest cells in mitosis. Flow cytometry analysis was performed on PBMCs fixed with 4% paraformaldehyde (PFA), permeabilized with 0.5% Triton X-100–PBS, and stained with anti-MPM2 (Millipore, 05-368). Cells were washed with PBS and incubated with a secondary antibody conjugated to Alexa Fluor 488 (Molecular Probes). Secondary antibody was washed with PBS, and DNA was stained with DAPI (2 mg/ml) for 30 min at 37°C.
8
Immunoprecipitation and Western Blot Analysis
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Cells were lysed in cell lysis buffer (50 mM Tris-HCl, pH7.5, 120 mM NaCl, 1 mM EDTA, and 1% NP-40) containing protease inhibitor cocktail (Roche, Indianapolis, IN, USA) and phosphatase inhibitor cocktail 2 (Sigma). Cellular lysates (1 mg) were immunoprecipitated, using the anti-CDC20 (5 μl, AR12, Millipore, Burlington, MA, USA) or anti-MAD2 (5 μg, A300, Bethyl Lab, Montgomery, TX, USA) antibodies. The immunoprecipitated products or total lysates were resolved on SDS polyacrylamide gel electrophoresis and transferred to PVDF membranes (BioRad). Proteins were detected with specific antibodies. The following antibodies were used in western blot analyses: anti-Cyclin B1 (H433), anti- CDC2 [54 (link)], anti-β-Tubulin (H-235, Santa Cruz Biotechnology, Santa Cruz, CA, USA); ant-MAD2 [48 (link)], anti-phosphoserine/threonine (22A, BD Biosciences); anti-β-Actin (M2, Sigma); anti-cyclin A2 (BF683), anti-cleaved Caspase 3 (D175), anti-p-RPS6 (S235/236) (2211), anti-p-CDC2 (Y15), anti-RB (4H1), anti-p-RB (S780) (D59B7), anti-APC2 (12301, Cell Signaling Technology, Danvers, MA, USA); anti-MPM2 (Millipore); anti-CDC20 (A301, Bethyl Lab). Horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibodies (Cell Signaling Technology; GeneTex, Irvine, CA, USA) were used and proteins were visualized with an enhanced chemiluminescence system (Advansta, Menlo Park, CA, USA).
9
Cell Cycle Analysis by Flow Cytometry
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Cells were harvested and fixed in 70% ethanol for at least 2 h at −20°C before immunostaining and flow cytometry analysis. Combined analysis of DNA content (1% propidium Iodide, PI, Sigma-Aldrich) and Phospho-H3 (anti-P-H3 S10, Millipore, #06–570; 1/400) or DNA content, BrdU (anti-BrdU, Abcam, ab6326; 1/250) and MPM2 (anti-MPM2, Millipore, #05–368; 1/250) was performed as previously described (58 (link)).
10
Lysed Cell Protein Analysis Protocol
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Cells were washed once in PBS and then lysed in 141 mM Tris Base, 106 mM Tris HCl, 2% LDS, 1% Benzonase nuclease (E1014, Millipore), pH 8.5, at room temperature for 15 min and snap frozen in liquid nitrogen. The protein concentration was determined using Rapid Gold BCA Protein Assay Kit (Pierce). Cell lysates (20 µg) were run on NuPAGE 4-12% Bis-Tris protein gels (Invitrogen) using standard procedures, transferred to 0.2 µm polyvinylidene fluoride membranes and blocked in 5% milk in TBST (0.05% Tween 20 in Tris-buffered saline) for 1 h at room temperature before overnight incubation with primary antibodies. Membranes were washed thrice in TBST then incubated with a HRP conjugated secondary antibody for 1 h at room temperature in TBST with 5% milk, and signals were detected by chemiluminescence (Clarity ECL, Biorad). Antibodies used in immunoblotting were as follows: rabbit polyclonal anti-Vinculin (Cell Signaling Technology, #13901), anti-Haspin (Abcam, ab21686, lot 156753), and anti-H3T3ph (B8634; (9)) with anti-rabbit IgG HRP (Cell Signaling Technology, #7074) and mouse monoclonal anti-VRK1 (Abcam, ab171933), anti-VRK2 (Santa Cruz, sc-365199), anti-H3S10ph (Millipore, 05-806), anti--tubulin (B-5-1-2, Sigma, T5168) and anti-MPM2 (Millipore, 05-368) with anti-mouse IgG HRP (Cell Signaling Technology, #7076).
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