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plenti6.3-EFR3A-mCherry was generated by PCR amplification of EFR3A-mCherry fragment from pcDNA3.1 EFR3A-mCherry was then subcloned into plenti6.3. EFR3A cDNA (Genescript) was cloned into the N-terminus of the mCherry fragment of pcDNA3.1-mCherry. The EFR3A-mCherry fragment was then sub...
pcDNA3-miR-200a was derived from pLenti 4.1 Ex miR200b-200a-429. Western blotting assay Western blotting assay was performed as described previously.46 Briefly, cells were lysed in a buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2 mM Na3VO4, 5 mM NaF, 1% Triton X-...
cDNAs then underwent a LR clonase reaction into plenti-CMV-Destination vectors for stable cell line generation. siRNA Experiments siRNAs targeting mouse Tfeb (#L-050607-02-0010) and mouse Tfe3 (#L-054750-00-0010) were purchased from Dharmacon. 368T1 cells were transfected with 20nM of each ...
In most eukaryotes, centromere identity relies not on DNA sequence but on the presence of CENP-A-containing nucleosomes, which are formed with the canonical histones H2A, H2B, and H4 at the active centromeres. CENP-A nucleosomes bind to 171-bp α-satellite DNA and are required for active ...
pcDNA3-miR-200a was derived from pLenti 4.1 Ex miR200b-200a-429. Western blotting assay Western blotting assay was performed as described previously.46 Briefly, cells were lysed in a buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 2 mm Na3VO4, 5 mm NaF, 1%...
For rescue experiments, the pLenti-EF1a-BSD vector (Addgene) system was utilized. Lentiviruses were made in 293T cells and the viral supernatants were collected at 60 h post-transfection and passed through a 0.45-μm filter prior to infection. After infection, HeLa and Huh7 cells were ...
We packaged lentivirus using pLenti-CRISPRv2-gRNA2. Lenti-CRISPRv2-gRNA2 was able to significantly decrease Dnmt3a level in the dDG 10 days after infection (Fig. 5c; T10 = 2.27, P < 0.05, Fig. 5d). After fear conditioning on day 1, mice were divided into two groups with ...
Insert and pLentiRNAGuide_001 were linearised with the BsmBI restriction enzyme and ligated using T4 DNA ligase (NEB #M0202S). Primers and templates are provided in Supplementary Data 1, part C. Cloning of PiggyBac plasmids All piggyBac plasmids used in this study have been obtained with ...
pLentiCRISPR v2-NF1 clone 2 GenScript N/A pDONR225-NF1 A gift from the RAS Initiative at the Frederick National Laboratory for Cancer Research at NCI N/A pCL-FLAG Zheng et al., 2012 N/A pCL-FLAG-NF1 This study N/A pCL-FLAG-NF1-R2258∗ This study N/A pCL-FLAG-NF1-Y2285...