Full-size cDNAs of the corresponding genes were amplified by PCR using a set of corresponding primers (Table S1) from plasmids kindly provided by Dr. Nagata [41]. Following digestion with AscI/SpeI enzymes, cDNAs were ligated with the AscI/SpeI treated pLVTHM-Klf4 backbone [42]. To pack ...
Next-generation DNA plasmids are starting to show promise in clinical trials. In one trial, a CAR-T targeting BCMA that was made with a reduced-size plasmid was tested in patients with relapsed/refractory multiple myeloma. The overall response rate among 90 patients was 57% in those ...
5a). Next, we constructed a series of luciferase reporter plasmids containing different lengths of the VEGFR2 promoter, including pGL3 (control), pGL3-VEGFR2-40 (−40 to +121 bp), pGL3-VEGFR2-125 (−125 to +121 bp), pGL3-VEGFR2-280 (−280 to +121 bp), pGL3-VEGFR2-420 (...
Confocal maximum intensity projections of Zika virions (green) and hybrid vesicles (red) on cells (blue nuclei) as used forb, representative of three images; scale bars, 20 μm.d, Anti-ZIKV (MOI 0.25) activity of hybrid semen EVs and occupation of Axl determined by confocal microscopy vi...
Vector plasmids for trans-complementation were generated by directed mutagenesis using infusion (Takara) utilizing the primers HB155-HB160 to insert untagged genes into the pEF1-mRFP-NAT target vector. 6 μg of circular vectors were transfected into A. castellanii using Superfect (QIAGEN). Selecti...
Construction of expression plasmids Transcripts for expression of Kunitz domains in the prokaryotic expression vector pET32b were generated by PCR using primers that contain restriction enzyme recognition sites for EcoRI and NotI (Table 4). The PCR protocol was the same as for RT-PCR and as templ...
This effect was cell-autonomous because co-electroporation of AA and membrane-tagged red fluorescent protein (GAP-RFP) plasmids yielded many RFP-positive, but no GFP-positive, axons navigating correctly to the tectum (data not shown). However, CPEB1-GFP tends to form large discrete puncta in ...
2.2. E. coli strains and plasmids Escherichia coli strain BH110 (nfo::kanR [Δ(xthA-pncA)90 X::Tn10]), is an isogenic derivative of AB1157, was from the laboratory stock [32]. B. subtilis ExoA was PCR-amplified from genomic DNA provided by Dr. U. Mechold (Institut Pasteur, Paris...
DNA fragments encoding our proteins of interest were amplified with PCR using oligonucleotides synthesized by IDT (see Supplementary Table S1 for a list of plasmids and Supplementary Table S2 for a list of primers) and CloneAMP HiFi PCR premix. Constructs were cloned using the In-Fusion HD Cloni...
Fig. 7: K48–K63-branched Ub chains increase in response to p97 inhibition and at sites of DNA damage. a–c, Pulldowns from U2OS cell lysates using agarose-immobilized NbSL3.3Q and subsequent western blot analysis of input and elution fractions with indicated antibodies. Cells were treated ...