For miRNA assays, NIH3T3 cells were grown to 60–80% confluency on 24-well plates. Per well, 500 ng of FLAG-CSDE1 and its mutant constructs were transfected using JetPRIME reagent (Polyplus transfection) according to the manufacturer’s instructions. 24 h posttransfection, the cells were again transfected with 500 ng of the miRNA reporter constructs using the reagent. The cells were lysed 48 h posttransfection with 100 μl of 1× passive lysis buffer (Promega) and the reporter activity was measured using Luminoskan Ascent (Thermo Fisher Scientific). For tethering assays, HeLa and HEK293 cells were grown to 60–80% confluency on 24-well plates. Per well, 500 ng of λNHA-constructs were transfected using JetPRIME reagent (Polyplus transfection) according to the manufacturer’s instructions. 48 h posttransfection, the cells were again transfected with 50 ng (100 ng for experiments made with HEK293 cells) each of Renilla and Firefly luciferase reporter constructs using the reagent. The cells were lysed 24 h posttransfection with 100 μl of 1× passive lysis buffer (Promega), and the reporter activity was measured using Luminoskan Ascent.
Jetprime reagent
Manufactured by Polyplus Transfection
Sourced in France, United States, China
JetPRIME is a cationic polymer-based transfection reagent used for the efficient delivery of nucleic acids, such as plasmid DNA, siRNA, and mRNA, into a variety of cell types. It is designed to form stable complexes with nucleic acids, facilitating their uptake and expression in the target cells.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (25 mentions)
Dual luciferase reporter assay system, by Promega (15 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (13 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (11 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (10 mentions)
Streptomycin, by Thermo Fisher Scientific (10 mentions)
Prl tk, by Promega (9 mentions)
Penicillin, by Thermo Fisher Scientific (9 mentions)
Pspax2, by Addgene (8 mentions)
Jetprime buffer, by Polyplus Transfection (7 mentions)
Pmd2 g, by Addgene (7 mentions)
Luciferase assay system kit, by Promega (5 mentions)
Hek293t cell, by American Type Culture Collection (5 mentions)
Interferin, by Polyplus Transfection (5 mentions)
Fetal bovine serum (fbs), by Merck Group (5 mentions)
Pmir report construct, by Thermo Fisher Scientific (4 mentions)
Fugene hd, by Promega (4 mentions)
Interferin reagent, by Polyplus Transfection (4 mentions)
Polybrene, by Merck Group (4 mentions)
Fetal bovine serum (fbs), by Sartorius (4 mentions)
380 protocols using jetprime reagent
1
miRNA and Tethering Assays in Mammalian Cells
2
Dual-Light System Reporter Assay
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Reporter gene assays were performed using the Dual-Light System (Applied Biosystems). ∼50% confluent HEK-293 T were transfected with SMARTpool siRNAs (12 pmoles cm−2) in six-well cell culture plates (Falcon) using jetPRIME reagent (Polyplus Transfection) following the manufacturer's instructions. Twenty-four hours after siRNA transfection, TEAD reporter plasmid (8xGTIIC–LUX56 (link); a gift from Stefano Piccolo, University of Padova, Italy; Addgene #34615) was transfected into the cells (200 ng cm−2) together with β-galactosidase plasmid (pEF-1α-LacZ 200 ng cm−2, Addgene #17430; kindly provided by Dr Shukry Habib, CSCRM, King's College London, UK) to normalize for transfection efficiency, using jetPRIME reagent (Polyplus Transfection) following the manufacturer's instructions. At 24 h post-plasmid transfection, cells were trypsinized and re-plated (1 × 104 cells) in triplicates into a 96-well plate, and allowed to grow for a further 24 h. Relative luciferase units were measured on a Glo-Max-Multi+ Multimode Reader (Promega) and normalized against β-galactosidase activity.
3
UGCG Overexpression and Knockdown in A375P Cells
4
Knockdown of JIP Proteins Using siRNA and shRNA
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The MKK4 (L-003574-00-0005), ZAK (L-005068-00-0005) JIP1 (L-003595-00-0005), JIP2 (L-012462-00-0005), JIP3 (L-003596-00-0005), JIP4 (L-017462-00-0005), and Negative Control (D-001810-10-05) ONTARGET plus smartpool siRNAs were from Thermo-Fisher. The MKK7 SilencerSelect siRNA (s11184) was from Ambion. Transfection of siRNA was performed using the jetPRIME reagent (Polyplus Transfection) according to manufacturer's instructions. The pLKO control shRNA and pLKO ZAK shRNA (Clone name: NM_016653.x-362s1c1) plasmids were a kind gift from Prof William Gallagher (Conway Institute, University College Dublin). Plasmid transfection was performed using the jetPRIME reagent (Polyplus Transfection) according to manufacturer's instructions, and stable cell line selection performed with puromycin (2 μg/mL).
5
CD44 Knockdown Using Targeted siRNA
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The expression of CD44 was downregulated using pre-designed target-specific small interfering RNA (siRNAs). Cells were transfected with siRNA by using the jetPRIME reagent (Polyplus-Transfection SA). The control siRNA (sictr, siN05815122147) and CD44 siRNA (siCD44, siG000000960B) were purchased from Guangzhou RiboBio Co., Ltd. The target sequence of CD44 siRNA was 5′-CCG CTT TGC AGG TGT ATT C-3′. By using the Basic Local Alignment Search Tool (BLAST) at NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi ), the targeted sequence was found to match well and specifically with both standard and variant forms (transcript variant 1-8, x1-x19) of CD44, suggesting that the designed siCD44 can knock down CD44 and its variant forms. The transfection efficiency was evaluated by RT-qPCR and western blot analysis. A total of 50 nM siCD44 or sictr mixed with jetPRIME reagent (Polyplus-Transfection SA) was added to the cells for 24 h, following which the solution was replaced with normal culture medium. Follow-up experiments were conducted 48 h after transfection.
6
Retroviral and Lentiviral Transduction Protocols
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Molecules expressed in NIH 3T3 and Raji cells were introduced by retrovirus transduction. Retrovirus was produced in Phoenix-ampho cells transfected with pCMV-VSV-G and MSCV-YFP containing the gene of interest using jetPRIME reagents (Polyplus Transfection).
Molecules expressed in A427, Jurkat, and K562 were introduced by lentiviral transduction. Lentivirus was produced in HEK293T cells transfected with pCMVR8.74, pCMV-VSV-G, and a lentiviral backbone vector containing the gene of interest using jetPRIME reagents (Polyplus Transfection). Virus-containing supernatant was harvested 48–72 hours after transfection and filtered through a 0.45 μm filter. Cells were spin-infected at 2000g for 120 minutes at 37°C in the presence of 5 μg/mL Polybrene (Merck Millipore) and 1–2 mL virus supernatant. Transduced cells were sorted using a BD FACSAria Fusion Cell Sorter (BD Biosciences).
Molecules expressed in A427, Jurkat, and K562 were introduced by lentiviral transduction. Lentivirus was produced in HEK293T cells transfected with pCMVR8.74, pCMV-VSV-G, and a lentiviral backbone vector containing the gene of interest using jetPRIME reagents (Polyplus Transfection). Virus-containing supernatant was harvested 48–72 hours after transfection and filtered through a 0.45 μm filter. Cells were spin-infected at 2000g for 120 minutes at 37°C in the presence of 5 μg/mL Polybrene (Merck Millipore) and 1–2 mL virus supernatant. Transduced cells were sorted using a BD FACSAria Fusion Cell Sorter (BD Biosciences).
7
Affinity Purification of Tagged Proteins from Mammalian Cells
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Phb1-KO hepatocytes were cotransfected with 2μg of His6-Nedd8 and FLAG-LKB1 or HA-Akt constructs using jetPRIMETM reagent (Polyplus). Cells were lysed in 6 M guanidinium-HCl, 0.1 M Na2HPO4/NaH2PO4, 0.01 M Tris-HCl pH 8, plus 10 mM β-mercaptoethanol and 5mM Imidazole. Lysates were mixed with 70 μl of low density Ni2+-NTA-agarose beads (ABT) precoated with BSA and prewashed with lysis buffer. Lysates were incubated with the beads for 3 hours at RT, successively washed first with lysis buffer, then twice with 8 M urea, 0.1 M Na2HPO4/NaH2PO4, 0.01 M Tris-HCl pH 8 plus 10 mM β-mercaptoethanol, and finally thrice with 8 M urea, 0.1 M Na2HPO4/NaH2PO4, 0.01 M Tris-HCl pH 6.3 plus 10 mM β-mercaptoethanol. After last wash, the beads were eluted with 200 mM Imidazole in 5% SDS, 0.15 M Tris-HCl pH 6.7, 30% glycerol, 0.72 M β-mercaptoethanol. The eluates were subjected to SDS-PAGE and the proteins transferred to a nitrocellulose membrane for Western blotting against FLAG (Sigma) and HA (Covance) antibodies.
8
Hepatocyte Transfection for LKB1 and Akt Studies
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Phb1-KO hepatocytes were transfected with 2 μg of pcDNA3-FLAG-LKB1 (Addgene), pLNCX1-HA-Akt (kindly provided by Dr. Carracedo, CIC bioGUNE, Spain) or cysteine protease (NEDP1-V5) plasmids using jetPRIMETM reagent (Polyplus). Two μg of pcDNA3-LacZ (Invitrogen) and pLNCX1 plasmids (provided by Dr. Carracedo) were used as negative controls of LKB1 and Akt, respectively.
9
Overexpression of MCJ in Hepatocytes
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WT primary hepatocytes were transfected with 2 μg of pCMV6-MCJ using jetPRIMETM reagent (Polyplus). pcDNA3-LacZ (Invitrogen) was used as a negative control.
10
Phb1-KO Hepatocytes Transfection
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Primary Phb1-KO hepatocytes were transfected with 100 nM Nedd8, LKB1 and Akt siRNA (Qiagen) using jetPRIMETM reagent (Polyplus). Controls were transfected with an unrelated siRNA (Qiagen). Protein knockdown was confirmed by Western blotting. siRNA sequences are shown in Supplementary Table V .
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