SOD2 cDNA fragments derived from pBI-EGFP-MnSOD (#16612; Addgene, Cambridge, MA, USA) were cloned into the FLAG-pcDNA3 vector and designated as pcDNA3-Flag-SOD2. Caco-2 cells were transduced with pcDNA3-Flag-SOD2 and treated with polyphenols (10 μM) for 48 h. Proteins were extracted by NP-40 lysis buffer (0.5% NP-40, 5 mM ethylenediaminetetraacetic acid (EDTA), 2 mM Na3VO4, 10 mM Tris-Cl pH 7.6, 150 mM NaCl, 5 μg/mL aprotinin, and 1 mM phenylmethylsulfonyl fluoride (PMSF)). SOD2 was immunoprecipitated with anti-Flag antibodies (M2; Sigma, St. Louis, MO, USA) and then immobilized with PureProteome Protein G Magnetic Beads (Merck Millipore, Billerica, MA, USA) overnight at 4°C. Immunoprecipitates were eluted with sample buffer (1 M Tris-HCl pH 6.8, 10% SDS, and 10% 2 ME).
Pbi egfp mnsod
Manufactured by Addgene
Sourced in United States
The PBI-EGFP-MnSOD is a plasmid that expresses a fusion protein of enhanced green fluorescent protein (EGFP) and manganese superoxide dismutase (MnSOD). The core function of this plasmid is to enable the expression and visualization of the MnSOD protein in cells.
Automatically generated - may contain errors
Lab products found in correlation
Quickchange 2 site directed mutagenesis kit, by Agilent Technologies (2 mentions)
Phr ig phr tripcmv ires egfp, by Addgene (2 mentions)
Transit 2020, by Mirus Bio (2 mentions)
Pureproteome protein g magnetic beads, by Merck Group (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
γh2ax, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
β tubulin, by Merck Group (1 mentions)
M0199, by Merck Group (1 mentions)
Mdivi, by Merck Group (1 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions)
5 protocols using pbi egfp mnsod
1
Overexpression of SOD2 in Caco-2 Cells
2
Engineered Plasmids and Viral Vectors
3
Constructing αSyn and ClpP Plasmids
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GFP-tagged αSyn-WT and -A53T plasmids (#40822, #40823), pBI-EGFP-MnSOD (#16612) were obtained from Addgene. To construct Myc-tagged αSyn-WT and –A53T, pCMV-Myc was digested with SalI and KpnI, and αSyn-WT or –A53T was PCR-amplified and inserted into the plasmid backbone. To construct Myc-tagged ClpP plasmid, pCMV-Myc was digested with EcoR1 and Xho1, and ClpP was PCR-amplified and inserted into the plasmid backbone. ClpP point mutant (S153A) was constructed using QuickChange II Site-Directed Mutagenesis Kit (200524, Agilent Technology). Cells were transfected with TransIT-2020 (Mirus Bio, LLC) following the manufacturer’s protocol.
To construct AAV-ClpP, human ClpP cDNA was inserted into the plasmid backbone AAV5.hSyn.eGFP.WPRE.bGH (Cat# AV-5-PV1696) which was obtained from Penn Vector Core, University of Pennsylvania. AAV-GFP-ClpP and AAV-GFP control were then packed to obtain AAVs in the same core facility.
To construct Lenti-ClpP, human ClpP cDNA was inserted into the plasmid backbone pHR-IG (pHR’tripCMV-IRES-eGFP) (53597, Addgene). Lenti-ClpP and lenti-control were then packed to obtain lentivirus for the infection as previously described [35 (link)].
To construct AAV-ClpP, human ClpP cDNA was inserted into the plasmid backbone AAV5.hSyn.eGFP.WPRE.bGH (Cat# AV-5-PV1696) which was obtained from Penn Vector Core, University of Pennsylvania. AAV-GFP-ClpP and AAV-GFP control were then packed to obtain AAVs in the same core facility.
To construct Lenti-ClpP, human ClpP cDNA was inserted into the plasmid backbone pHR-IG (pHR’tripCMV-IRES-eGFP) (53597, Addgene). Lenti-ClpP and lenti-control were then packed to obtain lentivirus for the infection as previously described [35 (link)].
4
Molecular Constructs for SOD2 and NME3 Studies
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The SOD2 expression vector, pAS3w.SOD2.bsd, was constructed by insertion of PCR products of pBI-EGFP-MnSOD, which was purchased from Addgene (plasmid #16612), at NheI (5′) and PmeI (3′) to pAS3w.bsd vector. Wild-type (WT) and N-terminal 30 amino acids deleted mutant (ΔN) of NME3-GFP expression vectors were constructed by insertion of PCR products amplified from NME3 cDNA at XhoI (5′) and HindIII (3′) to pEGFP-N1 vector. Tet-on expression vectors of Flag-NME3 were generated as described previously [4 (link)]. Mitochondrial Fusion promoter M-1 and Mdivi were purchased from Sigma-Aldrich (SML0629 for M-1, M0199 for Mdivi-1). Antibodies used in this study: γH2AX (Millipore, 05-636), Flag (Sigma-Aldrich, F3165), NME1/nm23-H1 (sc-343, Santa Cruz, Dallas, TX, USA), NME6 (GTX128818, Genetex, Irvine, CA, USA.), 8-oxoG/8-hydroxy-guanosine (ab62623, Abcam, Cambridge, UK), SOD2 (Millipore, 06-984), COX4 (4850S, Cell Signaling, Danvers, MA, USA), ATF4 (Cell Signaling, 11815S), MFN1 (Cell Signaling, 14739S), MFN2 (Abcam, ab56889), DRP1 (Cell Signaling, 8570S), SMCR7/Mid49 (16413-1-AP, Proteintech, Rosemont, IL, USA), SMCR7L/Mid51 (Proteintech, 20164-1-AP), β-tubulin (Sigma-Aldrich, T4026), β-actin (Sigma-Aldrich, A5441).
5
Stable and Transient SOD2 Modulation
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Lipofectamine® RNAiMAX reagent (Invitrogen) and LTX with Plus™ reagent (Invitrogen) were used following manufacturer’s protocol for transient transfection of SOD2 siRNA (S13268, Ambion, Austin, TX, USA) and pBI-EGFP-MnSOD (#16612, Addgene, Cambridge, MA, USA), pBI-EGFP (kindly provided by Dr. Hsiao-Sheng Liu, National Cheng Kung University, Taiwan), respectively. For stable knockdown, the cells were infected with SOD2-lentiviral short hairpin RNA (shRNA) or empty vector (both from RNAi Core, Academia Sinica, Taiwan). The next day, the infected cells were then selected for stable clones in antibiotic-containing medium for weeks, which was followed by confirmation of the knock-down efficiency and selection (Additional file 1 : Figure S1B).
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