Cells were lysed in NT2 buffer containing 50 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 1 mmol/L MgCl2, 0.05% Nonidet P-40, 1 mmol/L sodium orthovanadate, 0.5 mmol/L dithiothreitol, 1 mmol/L phenylmethylsulfonyl fluoride, 2 μg/mL aprotinin, 2 μg/mL leupeptin, and 2 μg/mL pepstatin. Proteins from cell lysates were separated by 10% SDS-PAGE gel electrophoresis (Bio-Rad), and transferred to nitrocellulose membranes (Invitrogen). Immunoblots were probed with antibodies recognizing: Aiolos (Cell Signaling Technology), Ikaros (Millipore), IRF7 (Cell Signaling Technology), DDX58 (Thermo Scientific), IFIT3 (Novus Biologicals), c-myc (Abcam), IRF4 (Santa Cruz), p65 (Cell Signaling Technology), p-p65 Ser236 (Cell Signaling Technology), p105/p50 (Cell Signaling Technology), p100/p52 (Cell Signaling Technology), Tubulin (Cell Signaling Technology), TBP (Cell Signaling Technology), and β-actin (Li-Cor). Signals were detected with a Li-Cor Odyssey imager or WES (Protein Simple).
Ddx58
Manufactured by Thermo Fisher Scientific
DDX58 is a laboratory equipment product manufactured by Thermo Fisher Scientific. It serves as a sensor that detects the presence of double-stranded RNA, which is a molecular pattern associated with viral infection. The core function of the DDX58 is to facilitate the recognition of this molecular pattern.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by LI COR (1 mentions)
Ikaros, by Merck Group (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Sds page gel electrophoresis, by Bio-Rad (1 mentions)
Ifit3, by Novus Biologicals (1 mentions)
Aiolos, by Cell Signaling Technology (1 mentions)
C myc, by Abcam (1 mentions)
Isg15, by Thermo Fisher Scientific (1 mentions)
Ifna4, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (1 mentions)
Ifna1, by Thermo Fisher Scientific (1 mentions)
Cxcl10, by Thermo Fisher Scientific (1 mentions)
Cxcl9, by Thermo Fisher Scientific (1 mentions)
Ifit1, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
2 protocols using ddx58
1
Immunoblot Analysis of Cellular Signaling
2
Quantitative PCR analysis of human liver
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Whole liver RNA was isolated using RNeasy mini kit (Qiagen, Hilden, Germany) including DNAse treatment according to manufacturer’s protocol starting with homogenization of liver tissue in RLT buffer. cDNA was generated by using an iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s protocol. Human specific gene expression was measured using Taqman primer/probe quantitative PCR, in TaqMan® Gene Expression Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). Primer/probe combinations were purchased from Thermo Fisher Scientific; CXCL10 (Hs01124251_g1), CXCL9 (Hs00171065_m1), DDX58 (Hs01061436_m1), GAPDH (Hs00266705_g1), IFIT1 (Hs01911452_s1), ISG15 (Hs01921425_s1), IFNA1 (Hs00855471_g1), IFNA4 (Hs01681284_sh), IFNB1 (Hs01077958_s1), MX1 (Hs00895608_m1), OAS1 (Hs00973637_m1), RSAD2 (Hs00369813_m1), STAT1 (Hs01013996_m1), TLR3 (Hs01551078_m1). Expression of target genes was normalized to the expression of GAPDH using the formula 2−ΔCt, ΔCt = Cttarget−CtGADPH. cDNA from non-chimeric mouse livers was used as control to test cross-reactivity of housekeeping and target genes. Due to the difference in hepatocyte donor baseline expression levels of examined genes (Suppl. Fig. 1 ), fold changes of transcripts were calculated to those of non-infected humanized livers from mice transplanted with the identical hepatocyte donor.
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