shRNAs targeting human NBR2 (NM_005821.2-615s1c1, NM_005821.2-514s1c1) were purchased from Sigma (Note that these two shRNAs target splicing isoforms #1 and #3 of NBR2, while can still achieve good knockdown efficiency when measured by real time PCR primer set designed to detect all three splicing isoforms of NBR2). siRNA targeting AMPKα were purchased from Origene (SR303721, SR303722). All three splicing isoforms of NBR2 were obtained from Thermo Fisher Scientific (MGC human NBR2 sequence-verified cDNAs, clone ID: 6452095, 4339497, 4826858) and then were subcloned into Lentiviral vector pLVX (Clontech). AMPK α, AMPK β and AMPK γ entry plasmids were obtained from Human ORFeome V5.1 library. The entry clones were subsequently recombined into gateway-compatible destination expression vectors with Flag tag through LR Gateway Technology (Invitrogen). cDNA corresponding to 1–312 a.a. of AMPK α1 was cloned into entry vector, and was subsequently recombined into gateway-compatible destination expression vectors with V5 tag through LR Gateway Technology (Invitrogen). Active human AMPK α2 protein and active human AMPK α1+AMPK β1+AMPK γ1 protein were purchase form Abcam (ab79803, ab126916). 2-Deoxy-D-glucose and compound C were purchased from Sigma (D6134, P5499). A-769662 was purchased from LC laboratories (A-1803).
Lr gateway technology
Manufactured by Thermo Fisher Scientific
The LR Gateway Technology is a molecular biology tool that facilitates the rapid and efficient transfer of DNA sequences between different vectors. It operates based on the site-specific recombination system of the bacteriophage lambda, allowing for the seamless movement of genetic material between compatible entry and destination vectors.
Automatically generated - may contain errors
Lab products found in correlation
Plvx lentiviral vector, by Takara Bio (2 mentions)
Ab79803, by Abcam (2 mentions)
Nm 005821.2 615s1c1, by Merck Group (2 mentions)
Nm 005821.2 514s1c1, by Merck Group (2 mentions)
Mgc human nbr2 sequence verified cdnas, by Takara Bio (2 mentions)
2 deoxy d glucose and compound c, by Merck Group (2 mentions)
A 769662, by LC Laboratories (2 mentions)
Fluorescent microscope, by Leica (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Pcr8 gw topo vector, by Thermo Fisher Scientific (1 mentions)
I max 2 dna polymerase, by iNtRON Biotechnology (1 mentions)
Isolate 2 pcr and gel kit, by Meridian Bioscience (1 mentions)
Mz 12 dissecting scope, by Leica (1 mentions)
Pdonr223 sars cov 2 nsp14, by Addgene (1 mentions)
5 protocols using lr gateway technology
1
Studying AMPK Regulation via NBR2 Knockdown
2
Studying AMPK Regulation via NBR2 Knockdown
3
Molecular Cloning of MtRbohE in M. truncatula
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A 344-bp cDNA fragment of 3'-UTR of MtRbohE (Mt4.0, Medtr8g095520.1) was amplified by PCR using the following primers: forward 5'-TGAGGATAACAGTGGAAGG-3' and reverse primer 5'-TCTCCTGGGACGACTATAA-3' and cloned into the pDNOR207 vector using the BP Gateway technology (Invitrogen). The resulting vector was recombined with the pK7GWIWG2D(II) vector (Karimi et al. 2002) using the LR Gateway technology (Invitrogen), according to the manufacturer's recommendations. The construct was checked by DNA sequencing introduced by electroporation into Agrobacterium rhizogenes strain ARqua1, and used for M. truncatula root transformation as previously described (Medicago handbook). The pK7GWIWG2D(II) empty vector has been used as control. After 2 weeks, transgenic roots were selected under fluorescent microscope (Leica), and transferred on SHb10 medium (Medicago handbook) supplemented with 200 mg L -1 augmentin (amoxicillin:clavulanic acid [5:1]) and 20 mg L -1 kanamycin (Sigma-Aldrich). Root cultures were keept at 20°C in the dark and subcultured each 3 weeks on new medium. After 3 subculturing, augmentin was removed and the level of MtRbohE was evaluated by qRT-PCR (Fig. S2).
4
Enhancer Cloning for Zebrafish Transgenesis
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For enhancer cloning, zebrafish and human genomic fragments containing the studied CREs were amplified with iMAX-II DNA Polymerase (Intron Biotechnology) using primers from Supplementary Table S1 . The PCR fragments were purified using Isolate II PCR and Gel Kit (BIOLINE), sub-cloned in the pCR8/GW/TOPO vector (Invitrogen) and then transferred, through recombination using Gateway LR technology (Invitrogen), to the enhancer detection vector for zebrafish transgenesis, containing the strong midbrain enhancer z48 and the green fluorescent protein (GFP) reporter gene under the control of the gata2 minimal promoter (Gehrke et al., 2015 (link)).
Zebrafish transgenic embryos were generated using the Tol2 method (Kawakami et al., 2004 (link)). One-cell-stage embryos were injected with 3–5 nl of a solution containing 30 ng/μl of Tol2 mRNA, 20 ng/μl of phenol:chloroform-purified enhancer detection vector, and 0.05% of phenol red solution. Injected embryos (F0) were selected for GFP expression at 48 hpf, raised to sexual maturity and screened for germline transmission. GFP-expressing F1 embryos were photographed at 24 and 48 hpf stages with a digital CCD camera (MagnaWre, Optronix) mounted on an MZ-12 dissecting scope (Leica). Three independent stable transgenic lines showing similar GFP expression patterns were generated.
Zebrafish transgenic embryos were generated using the Tol2 method (Kawakami et al., 2004 (link)). One-cell-stage embryos were injected with 3–5 nl of a solution containing 30 ng/μl of Tol2 mRNA, 20 ng/μl of phenol:chloroform-purified enhancer detection vector, and 0.05% of phenol red solution. Injected embryos (F0) were selected for GFP expression at 48 hpf, raised to sexual maturity and screened for germline transmission. GFP-expressing F1 embryos were photographed at 24 and 48 hpf stages with a digital CCD camera (MagnaWre, Optronix) mounted on an MZ-12 dissecting scope (Leica). Three independent stable transgenic lines showing similar GFP expression patterns were generated.
5
SARS-CoV-2 NSP10 and NSP14 Cloning
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pDONR223 SARS-CoV-2 NSP10 (Addgene; plasmid no. 141264; http://n2t.net/addgene:141264 ; Research Resource Identifier: Addgene_141264) and pDONR223 SARS-CoV-2 NSP14 (Addgene; plasmid no. 141267; http://n2t.net/addgene:1412647 ; Research Resource Identifier: Addgene_141267); both were a kind gift from Fritz Roth (34 (link)). Using Gateway LR technology (Invitrogen), NSP10 and NSP14 were subcloned individually into gateway competent expression vector pDEST527 (N-terminal His-tags NSP10 and NSP14) for bacterial expression.
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