Mouse LUZP1 full‐length cDNA was inserted into a pFastBac‐based plasmid that was constructed by inserting a GST coding sequence and a multi‐cloning site from the pGEX6P2 vector (#28‐9546‐50; GE Healthcare) into pFastBac1 vector (#10712024; Invitrogen). This plasmid was transformed into DH10Bac E. coli (#10361012; Invitrogen) to obtain a bacmid. Insect Sf9 cells were then transfected with the bacmid using Cellfectin II (#10362100; Invitrogen). After 3 days of transfection, the culture medium containing baculovirus was collected and added to other Sf9 cells in 6‐well dishes for infection. After 3 days of infection, Sf9 cells were lysed with RIPA buffer (100 μl/well). The cell lysate, clarified by centrifugation at 20,400 g at 4°C for 30 min, was incubated with 500 μl glutathione Sepharose 4B (#17‐5132; GE Healthcare) at 4°C for 120 min. After five washes with RIPA buffer, beads were eluted with 10 mM glutathione in 50 mM Tris buffer (pH 8.0). Protein solutions were dialyzed in cellulose dialysis membrane soaked in ice‐cold HBS to remove glutathione at 4°C overnight.
Cellfectin 2
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Cellfectin II is a lipid-based transfection reagent designed for the efficient delivery of nucleic acids into eukaryotic cells. It facilitates the uptake of plasmid DNA, siRNA, and other nucleic acid molecules into the cells, enabling researchers to study gene expression, knockdown, or other cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Dh10bac e coli, by Thermo Fisher Scientific (6 mentions)
Sf9 cells, by Thermo Fisher Scientific (5 mentions)
Bac to bac expression system, by Thermo Fisher Scientific (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Dansylcadaverine, by Merck Group (3 mentions)
Tetracycline, by Merck Group (3 mentions)
Coomassie brilliant blue, by Merck Group (3 mentions)
Kanamycin, by Merck Group (3 mentions)
Laemmli buffer, by Merck Group (3 mentions)
X gal, by Thermo Fisher Scientific (3 mentions)
Culture mediums, by Thermo Fisher Scientific (3 mentions)
Recombinant human transglutaminase 2 tgm2, by R&D Systems (3 mentions)
Anti tg2 antibody, by Abcam (3 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Thermo Fisher Scientific (3 mentions)
N n dimethylcasein, by Merck Group (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Dithiothreitol (dtt), by Merck Group (3 mentions)
3 3 diaminobenzidine (dab), by Merck Group (3 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (3 mentions)
Protease inhibitor, by Merck Group (3 mentions)
97 protocols using cellfectin 2
1
Recombinant GST-LUZP1 Protein Production
2
Hippo Pathway Regulation in S2 Cells
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S2 cells cultured at 25°C were transfected with Actin-Gal4, pIE1-4-myc-Hippo, pIE1-4-myc-Warts, pIE1-4-HA-Merlin and UAS-FLAG-His6-Rae1, pAc5.1-His6-FLAGx3-Rae1, pAc5.1-Rae1-V5-His using Cellfectin II (Invitrogen) or Effectene (Qiagen). HEK-293T, U87MG and HeLa cells were cultured in DMEM (Invitrogen) containing 10% FBS (Gemini) and 50 μg/mL penicillin/streptomycin (Gemini). Transfection with pCMV5-FLAG-Mst1, pCMV2-FLAG2 Lats1, pCMV-FLAG Yap2 S127A, pCMV-FLAG-Yap2 5SA, using Effectene (Qiagen) was performed according to the manufacturer's instructions.
3
Culturing and Transfecting Insect and Mammalian Cells
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Drosophila Schneider S2 cells were cultured in SFX-Insect medium (Hyclone, USA) at 27 °C without CO2 and were split at the density of ~6 × 106 cells/ml every 3 days. 293 T, HeLa, TZM-bl and COS-1 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS (fetal bovine serum), penicillin (100 U/ml) and streptomycin (100 μg/ml), in a humidified atmosphere containing 5% CO2 at 37 °C. Drosophila S2 cells and the mammalian cells were transfected using reagents Cellfectin II and Lipofectamine 2000 (Invitrogen, USA) respectively following the manufacturer’s recommendations.
4
Baculovirus Expression System Clone Selection
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Bacmid DNA and Cellfectin II (Invitrogen) were poured into two separate FACS tubes containing Sf900 III medium (Gibco), and were incubated for 15 min at room temperature. The two tubes were then mixed, incubated for a further 15 min, and added to one well of a six-well plate with one million Sf9 cells (Invitrogen). After 5 days, lysis plaques had been performed, and the stability of five clones was studied using quantitative polymerase chain reaction (qPCR) and Western blot. Two of the five clones were then amplified in a 125 mL shake flask (Corning) with a working volume of 75 mL. Baculovirus titrations were performed using the lysis plaque technique.19
5
Recombinant Baculovirus Generation for T. gondii and Influenza
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rBVs expressing T. gondii IMC or influenza M1 were generated as described previously [11 (link)]. Transfection of DNA containing these genes was done using Cellfectin II (Invitrogen) with SF9 cells as recommended by the manufacturer, followed by transformation of pFastBac containing T. gondii IMC or M1 with white/blue screening. The rBVs were derived using a Bac-to-Bac expression system (Invitrogen) according to the manufacturer’s instructions.
6
Visualizing Baculovirus p26 Nuclear Localization
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In order to observe potential nuclear localization of p26, EGFP-p26 was expressed in BmN cells using the Bac-to-Bac expression system. The recombinant donor vector pFastBacHTb (Invitrogen, Shanghai, China) was reconstructed to contain egfp and p26, designated as pBacHT-EGFP-p26. The plasmid was transformed into BmDH10Bac E. coli (Invitrogen) to generate the recombinant bacmid, designated as EGFP-p26/rBmBac. The extracted EGFP-p26/rBmBac DNA was transfected into BmN cells by using Cellfectin II (Invitrogen). The recombinant virus was obtained and used to infect BmN cells, and fluorescence was directly viewed under a confocal laser scanning microscope at 24 and 48 hpi. EGFP/rBmBac-infected cells were used as the control.
7
Heterologous Expression of Slo1 Protein
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Heterologous expression of Slo1 was carried out using the BacMam method as previously described (22 (link), 24 (link)). In brief, bacmids were generated by transforming the ALFA-Slo1-HRV3C-GFP-1D4 construct into Escherichia coli DH10Bac cells. Baculoviruses were then produced by transfecting Sf9 cells with the bacmid using Cellfectin II (Invitrogen).
Baculoviruses, after two rounds of amplification, were used for cell transduction. Suspension cultures of HEK293S GnTI- cells were grown at 37 °C to a density of ~3 × 106 cells/mL and infected with 10% (v:v) of the baculovirus. After 20 h, 10 mM sodium butyrate was supplemented, and the temperature was shifted to 30 °C. Cells were harvested ~40 h after the temperature switch. For total membrane isolation, cell pellets were flash-frozen in liquid N2 and used later. For plasma membrane isolation, cell pellets were used immediately without freezing.
Baculoviruses, after two rounds of amplification, were used for cell transduction. Suspension cultures of HEK293S GnTI- cells were grown at 37 °C to a density of ~3 × 106 cells/mL and infected with 10% (v:v) of the baculovirus. After 20 h, 10 mM sodium butyrate was supplemented, and the temperature was shifted to 30 °C. Cells were harvested ~40 h after the temperature switch. For total membrane isolation, cell pellets were flash-frozen in liquid N2 and used later. For plasma membrane isolation, cell pellets were used immediately without freezing.
8
Expression and Purification of M-T7 Mutants
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M-T7 and M-T7 point mutations were expressed and purified as previously described. In brief, M-T7 mutants were generated by mutagenic PCR using M-T7pFastBacDualeGFP as the template112. Mutant constructs and wild type M-T7 were transformed into DH10Bac bacteria (Invitrogen.Carlsbad, CA), and blue/white screened on LB + Kan + Tet + Gen + IPTG+ X-gal plates. Bacmids were purified and used to transfect Sf9 insect cells with Cellfectin II (Invitrogen. Carlsbad, California). Baculovirus supernatants were collected to infect insect cells and express the various M-T7 mutant proteins. M-T7 and each of the three mutant constructs were then purified by sequential column purification as previously described19 ,29 (link).
9
Transglutaminase 2 Purification and Detection
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All restriction enzymes, X-gal, and IPTG were obtained from Fermentas (Lithuania). dNTP and Taq DNA polymerase were purchased from Roche (Germany). Culture mediums were purchased from Gibco (USA). Cellfectin II and TRIZOL were purchased from Invitrogen (USA). Coomassie brilliant blue, HCL, NaOH and boric acid were from Merck (Germany). Nitrocellulose membrane from Schleicher & Schuell BioScience (Germany). Gentamicin, Tetracycline, Kanamycin, Tris, SDS, Protease inhibitor, Laemmli buffer, DAB, dansylcadaverine, N,N dimethyl casein, EDTA, Cacl2 and DTT were obtained from Sigma (USA). Primers were synthesized by Pishgam biotech (Iran). cDNA synthesis were performed by Takara kit (Japan). Spodoptera frugiperda (Sf9) cells were from National Cell Bank of Iran (NCBI, Pasteur Institute of Iran). Recombinant human transglutaminase 2/TGM2 was purchased from R&D systems (USA). Anti-TG2 antibody was from Abcam (UK) and anti-rabbit IgG was obtained from Santa Cruz Biotechnology (USA).
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All restriction enzymes, X-gal, and IPTG were obtained from Fermentas (Lithuania). dNTP and Taq DNA polymerase were purchased from Roche (Germany). Culture mediums were purchased from Gibco (USA). Cellfectin II and TRIZOL were purchased from Invitrogen (USA). Coomassie brilliant blue, HCL, NaOH and boric acid were from Merck (Germany). Nitrocellulose membrane from Schleicher & Schuell BioScience (Germany). Gentamicin, Tetracycline, Kanamycin, Tris, SDS, Protease inhibitor, Laemmli buffer, DAB, dansylcadaverine, N,N dimethyl casein, EDTA, Cacl2 and DTT were obtained from Sigma (USA). Primers were synthesized by Pishgam biotech (Iran). cDNA synthesis were performed by Takara kit (Japan). Spodoptera frugiperda (Sf9) cells were from National Cell Bank of Iran (NCBI, Pasteur Institute of Iran). Recombinant human transglutaminase 2/TGM2 was purchased from R&D systems (USA). Anti-TG2 antibody was from Abcam (UK) and anti-rabbit IgG was obtained from Santa Cruz Biotechnology (USA).
10
Cloning and Transfection of Baculovirus Constructs
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PMD19-T-E0 + E2 and PMD19-T-E2 + E2 were digested by BamH I and EcoR I, and then ligated to the pFastBac1 vector (Invitrogen, United States) digested by the same enzyme. The correct clones were verified via Sanger sequencing and were, respectively, named pFastBac-E0 + E2 and pFastBac-E2 + E2, which were transformed into DH10Bac™ E. coli cells (Invitrogen, United States), and followed by the antibiotic selection. After Sanger sequencing and PCR verification, the correct clones were named recombinant rBacmids-E0 + E2 and rBacmids-E2 + E2, respectively. 1 × 106 Sf9 cells were transfected with 1 μg rBacmids-E0 + E2 or rBacmids-E2 + E2 plasmid using Cellfectin II (Invitrogen, United States) according to the manufacturer’s guidelines. After 5–7 days, cells and supernatants were collected, respectively, after obvious CPE appeared, and stored at –80°C.
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