To assess the diameters of the nuclei, phase-contrast images were used. Approximate nuclear volumes were calculated based on an assumption that the erythroblast’s nuclei have a spherical shape. Immunostaining of the cells and isolated nuclei were performed as previously described25. The following primary antibodies were used: mouse anti-PML (Millipore, MAB3738) and mouse anti-coilin (Abcam, ab87913). The primary antibodies bound to antigens were visualized using Alexa Fluor 488-conjugated secondary antibodies: rabbit anti-mouse AF488 (Invitrogen, A11059). The DNA was counterstained with 4’,6-diamidino-2-phenylindole (DAPI). The results were inspected using a Zeiss Axio Scope.A1 microscope equipped with a 100× EC Plan-Neofluar 100×/1.30 Oil-immersion objective.
Mab3738
Manufactured by Abcam
Sourced in United States
MAB3738 is an antibody product manufactured by Abcam. It is a monoclonal antibody that can be used in various laboratory applications. The core function of this product is to recognize and bind to a specific target, but the details of its intended use are not provided.
Automatically generated - may contain errors
Lab products found in correlation
Imagequant las 4000, by GE Healthcare (2 mentions)
Ab72137, by Abcam (2 mentions)
Ab87913, by Abcam (1 mentions)
Ag 20b 0014 c100, by Alomone (1 mentions)
Il 1β, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
β tubulin, by Cell Signaling Technology (1 mentions)
Nupage novex 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Caspase 1, by Novus Biologicals (1 mentions)
Las 4000, by Cytiva (1 mentions)
A2547, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose membrane, by Pall Corporation (1 mentions)
Ab32058, by Abcam (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Gold enhancement solution, by Nanoprobes (1 mentions)
Leo 512 electron microscope, by Zeiss (1 mentions)
Nanogold conjugated secondary antibody, by Nanoprobes (1 mentions)
Hrp labeled secondary antibody, by Thermo Fisher Scientific (1 mentions)
5 protocols using mab3738
1
Visualizing Erythroblast Nuclear Structure
2
Comprehensive Western Blot Analysis
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Total cell lysates from BMDM, pRMØs, PBMCs, B16-F10, and LL/2 cells were lysed in a buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, and 0.2% SDS supplemented with protease and phosphatase inhibitor cocktails. Protein extracts were quantified by the Lowry method, and 10 μg of proteins were loaded and separated on a NuPAGE Novex 4–12% Bis-Tris Gel (Life Technologies) and transferred onto a nitrocellulose membrane.
Primary antibodies were raised against human PML (Abcam, ab72137), mouse PML (Millipore, MAB3738), mouse PML (Abcam, #67761), the IL-1 receptor (I/IL-1R1, Abcam, ab107270), Sigma-1R (Sigma-Aldrich, HPA018002), IP3R3 (BD Biosciences, 610312), GAPDH (Cell Signaling, #2118), VDAC (Abcam, ab15895), β-tubulin (Cell Signaling, #55685), NLRP3 (Adipogen, AG-20B-0014-C100), P2X7 (Alomone, APR-004), P2X7 (Sigma-Aldrich, P8232), Caspase-1 (Novus Biological, #14F468), IL-1β (Cell Signaling Technology, #12242), and β-Actin (Sigma-Aldrich, #A2668). HRP-conjugated antibodies (#31430; #31460. WB, 1:5,000 from Thermo Fisher Scientific) were used, followed by detection using ImageQuant LAS 4000 (GE Healthcare) (see Supplemental Material). The western blots shown in the figures are representative of at least 3 independent experiments.
Primary antibodies were raised against human PML (Abcam, ab72137), mouse PML (Millipore, MAB3738), mouse PML (Abcam, #67761), the IL-1 receptor (I/IL-1R1, Abcam, ab107270), Sigma-1R (Sigma-Aldrich, HPA018002), IP3R3 (BD Biosciences, 610312), GAPDH (Cell Signaling, #2118), VDAC (Abcam, ab15895), β-tubulin (Cell Signaling, #55685), NLRP3 (Adipogen, AG-20B-0014-C100), P2X7 (Alomone, APR-004), P2X7 (Sigma-Aldrich, P8232), Caspase-1 (Novus Biological, #14F468), IL-1β (Cell Signaling Technology, #12242), and β-Actin (Sigma-Aldrich, #A2668). HRP-conjugated antibodies (#31430; #31460. WB, 1:5,000 from Thermo Fisher Scientific) were used, followed by detection using ImageQuant LAS 4000 (GE Healthcare) (see Supplemental Material). The western blots shown in the figures are representative of at least 3 independent experiments.
3
Co-Immunoprecipitation of Protein Complexes
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Co-IP assays require lysing of the cellular samples using RIPA lysis buffer containing a protease inhibitor cocktail. Whole-cell lysates were then treated with desired antibodies. Afterward, magnetized protein A/G beads (MedChem Express, USA, #HY-K0202) were used to capture the target protein. SDS-PAGE was used to separate immunoprecipitates or whole-cell lysates, followed by transfer to nitrocellulose membranes (Pall Corporation, Mexico, USA). The membranes were incubated overnight at 4°C with the following primary antibodies: PML (1:1000; Merck Millipore, USA, #MAB3738), SUMO-1 (1:500; Abcam, USA, #ab32058), SUMO-2/3(1:500; Abcam, USA, #ab3742), p53 (1:1000; Proteintech, USA, #10442-1-AP), p-p53 (1:1000; Cell Signaling Technology, USA, #9284) and α-SMA (1:1000; Sigma, USA, #A2547), Ubiquitin (1:500; Santa Cruz Biotechnology, USA, #sc-8017), Alpha Tubulin (1:2000; Proteintech, USA, #66031l-1-lg) and GAPDH (1:10000; ABclonal, USA, #AC002). After one-hour incubation with the appropriate secondary antibodies, the membranes were scanned with the Odyssey infrared imaging system (LI-COR, Lincoln, NE, USA). The intensity of bands was quantified using Image-J software.
4
Ultrastructural Immunogold Localization
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Cells were fixed with 4% formaldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 hr at room temperature. Subsequently, cells were permeabilized, blocked, and incubated sequentially with PML primary antibodies (Millipore MAB3738 or Abcam ab72137) and nanogold-conjugated secondary antibodies (Nanoprobes) diluted in blocking buffer. After washes, cells were re-fixed in 1% glutaraldehyde, and nanogold was enlarged with gold enhancement solution (Nanoprobes). Cells were post-fixed with osmium tetroxide, embedded in epon, and processed into ultrathin slices. After contrasting with uranyl acetate and lead citrate, sections were analyzed with a Zeiss LEO 512 electron microscope.
5
Immunoblotting for PML protein
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For the immunoblotting, the following primary antibodies were used: mouse anti-PML [MAB3738] (1:1000) and rabbit anti-PML [ab72137] (1:1000) from Abcam. Finally, the membranes were incubated with the appropriate horseradish peroxidase (HRP)-labeled secondary antibodies (Thermo Fischer Scientific), followed by detection by chemiluminescence (Thermo Scientific) using Image Quant LAS4000 (GE Healthcare).
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