Total proteins were extracted using radio immunoprecipitation assay buffer containing 1% phenylmethanesulfonyl fluoride and quantified by a BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Equivalent amounts of protein were loaded onto 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred into polyvinylidene difluoride membranes. After blocked by skim milk, membranes were incubated in the primary antibodies overnight at 4°C. Primary antibodies were as follows: NDRG3 (1:1,000; Abcam), Src (1:500; Cell Signaling Technology, Boston, MA, USA), p-Src (Try419, 1:1,000; Santa Cruz Biotechnology Inc., Dallas, TX, USA), and GAPDH (1:10,000; Abcam). Membranes were then incubated with horseradish peroxidase-conjugated secondary antibody for 2 hours at room temperature and were visualized by a Tanon detection system with electrochemiluminescence reagent.
Ndrg3
Manufactured by Abcam
Sourced in United Kingdom
NDRG3 is a protein that plays a role in cellular processes. It is a member of the N-myc downstream-regulated gene (NDRG) family. NDRG3 is expressed in various tissues and is involved in cellular differentiation and stress response.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Abcam (2 mentions)
Hatf00010, by Merck Group (1 mentions)
Ndrg1, by Thermo Fisher Scientific (1 mentions)
Genetools of chemigenius, by Syngene (1 mentions)
Ne pertm nuclear and cytoplasmic extraction kit, by Thermo Fisher Scientific (1 mentions)
Ndrg2, by Abcam (1 mentions)
β actin antiserum, by Merck Group (1 mentions)
Maspin, by BD (1 mentions)
Hif 2α, by Novus Biologicals (1 mentions)
Hif 1α, by BD (1 mentions)
4 protocols using ndrg3
1
Quantitative Analysis of Protein Expression
2
Western Blot Protein Analysis
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3
Nuclear and Cytoplasmic Protein Extraction
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For nuclear and cytoplasmic extraction, cells were cultured in an RPMI-1640 medium with 10% FCS for 48 h, and then harvested with trypsin, and washed twice with PBS. Nuclear and cytoplasmic fractions were separated using the NE-PERTM Nuclear and cytoplasmic extraction kit (Thermo, Rockford, NJ, USA) as described by the manufacturer. Equal amounts of whole cell, nuclear, or cytoplasmic lysis were loaded onto a 10% SDS-polyacrylamide gel and assayed by enhanced chemiluminescence as described by the manufacturer (PerkinElmer Inc, Waltham, MA, USA). Blotting membranes were probed with antiserum of MT3 (Sigma-Aldrich Co.), heme oxygenase-1 (HO-1; Stressgen, Victoria, BC, Canada), NDRG1 (Invitrogen), NDRG2, NDRG3 (Abcam, Cambridge, UK), HIF-1α, MASPIN (BD Biosciences, San Jose, CA, USA), HIF-2α (Novus, Littleton, CO, USA), or β-actin antiserum (Millipore, Billerica, MA, USA). The intensities of different bands were analyzed using the GeneTools of ChemiGenius (Syngene, Cambridge, UK).
4
Immunohistochemistry of NDRG3 Protein
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