Snap frozen maternal and fetal organs were homogenized in ice-cold radioimuno precipitation assay buffer (0.5 mol/L Tris-Cl, pH 7.4, 1.5 mol/L NaCl, 10 mmol/L ethylenediaminetetraacetic acid [EDTA], 2.5% deoxycholic acid, 10% NP-40, protease inhibitor, and phosphatase inhibitor), by rapid agitation in the presence of beads and spun at 13 000 g at 4 °C for 10 minutes. mTORC1 and mTORC2 signaling activity in maternal and fetal tissue homogenates was assessed by determining total protein expression and phosphorylation of key downstream targets using Western blot and commercial antibodies (Cell Signaling Technology, Boston, MA).
Western blot
Manufactured by Cell Signaling Technology
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Western blot is a widely used analytical technique in molecular biology and biochemistry. It is used to detect and quantify specific proteins within a complex mixture of proteins extracted from cells or tissues. The Western blot process involves the separation of proteins by size using gel electrophoresis, transfer of the separated proteins to a membrane, and the detection of the target protein using specific antibodies.
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Lab products found in correlation
Puromycin, by Cell Signaling Technology (2 mentions)
Puromycin, by Thermo Fisher Scientific (2 mentions)
Pcmv cat t7 sb100, by Addgene (2 mentions)
Anti sox2 d6d9, by Cell Signaling Technology (2 mentions)
Tracrrna atto 550, by Integrated DNA Technologies (2 mentions)
Custom crrnas, by Integrated DNA Technologies (2 mentions)
Silentfect lipid reagent for rnai, by Bio-Rad (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Phospho stat1 tyr701, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Lenticas9 blast, by Addgene (1 mentions)
6 protocols using western blot
1
Maternal-Fetal mTORC1/2 Signaling Assay
2
Modulation of IFN-α Signaling in Huh7.5 Cells
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Huh7.5 cells were seeded at 3 × 105/ml, 2 ml per well in 6-well plates in antibiotic-free medium for 24 hours before either 4 μg USP18 WT or 4 μg USP18 mutant form (USP18 C64S) was transfected into each well. 36 hours posttransfection, 10 U/ml IFNα was added to each well. The cells were harvested at 0 min, 30 min, 2 hours, 4 hours, 8 hours, and 24 hours posttreatment. Total protein was extracted using lysis buffer and 1 mM EDTA with protease inhibitor cocktail (Sigma). Phospho-STAT1 (Tyr701) and total STAT1 were detected by western blot (Cell Signaling, USA), and the band densities were analyzed using ImageJ software. At each time point, total RNA was extracted by TRIzol (Thermo Fisher Scientific, USA), and ISG mRNAs were determined by real-time PCR described above with the primers listed in Table 1 . All the primers were self-designed with the Primer3 program and were synthesized by a commercial company.
MicroRNA-122 expression levels were also determined using the microRNA-122 kit (Applied Biosystems, USA) following the manufacturer's protocols and normalized to U6.
MicroRNA-122 expression levels were also determined using the microRNA-122 kit (Applied Biosystems, USA) following the manufacturer's protocols and normalized to U6.
3
CRISPR-Cas9 Screening in MM1S and KMS18 Cells
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MM1S and KMS18 cells were first virally transduced with LentiCas9-Blast (Addgene #52962) and Cas9 expression was confirmed by Western Blot (Cell Signaling Technology #14697). Subsequently, Cas9-positive cells were virally transduced with subpools of the Avana library of single guide RNAs (sgRNAs) in triplicate (~1K cells/guide), and samples were drawn on day 4 and day 27 following infection. DNA was extracted using QIAGEN DNA micro kits and deep targeted sequencing of the sgRNAs was performed at the Massachusetts General Hospital Center for Computational & Integrative Biology DNA Core. Following deconvolution and barcode quantitation with PoolQ, read counts were normalized for total depth, multiplied by 10^6, and log2-transformed with a pseudocount of 1. Replicates with < 200K reads or correlation < 0.7 were discarded. The remaining technical replicates per subpool were collapsed by computing their median. Log2 fold-changes were computed using day 4 as the baseline estimate and normalized by subtracting the median log2 fold-change of all non-targeting sgRNAs in the respective subpool. A final guide-level matrix was obtained by computing the median log2 fold-change across subpools. Copy number-corrected gene-level matrices were obtained using Ceres following z-mad normalization, as described previously, and used for downstream analyses57 (link).
4
Knockdown of IDO-1 in LN-18 Cells
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Example 15
LN-18 cells were infected with an MOI of 0.1 using the RRV vector expressing the IDOshRNA shown in
5
CRISPR/Cas9-Mediated SOX2 Knockout in CWR-R1 Cells
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To generate CRISPR/Cas9-mediated SOX2KO cell lines, parental CWR-R1 cells were co-transfected with pT2-EF1a-Cas9-P2A-puro and pCMV(CAT)T7-SB100 (#34879, Addgene; Watertown, MA) using Lipofectamine 2000 (Invitrogen) to introduce Cas9 expression. Stable constitutive Cas9 expression was accomplished by SB100 transposase integration of EF1a-Cas9-P2A-puro. Cas9-expressing cells were selected for and maintained with puromycin (1 mg/mL, Invitrogen) 48 h after transfection. Constitutive Cas9 expression was confirmed by western blot (# 14697, Cell Signaling Technologies) after 1 week of puromycin selection. Two custom crRNAs (Integrated DNA Technologies; Coralville, IA) targeting the N-terminus of SOX2 were selected using CHOPCHOP software (https://chopchop.cbu.uib.no ) (SOX2 crRNA #1: 5’-CGGGCCCGCAGCAAACTTCG-3’, SOX2 crRNA #2: 5’-CGCCCGCATGTACAACATGA-3’) and were individually complexed with tracrRNA-ATTO 550 (#1075927, Integrated DNA Technologies) at a 1:1 ratio immediately before transfection. A final concentration of 10 nM crRNA:tracrRNA duplexes were transfected into CWRR1-Cas9 cells using siLentFect Lipid Reagent for RNAi (#1703360, BioRad) following manufacturer guidelines. Limited dilution was performed to isolate three clonal knockout cell lines, and successful knockout of SOX2 was validated by western blot (anti-SOX2(D6D9), #3579, Cell Signaling Technologies).
6
CRISPR/Cas9-Mediated SOX2 Knockout in CWR-R1 Cells
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