Kinetic analyses of purified 3beta-HSD1 show that the Michaelis-Menten constants (Km) for substrates and cofactor are decreased dramatically (three- to eight-fold) by the addition of beta-mercaptoethanol (BME), which suggest that a disulfide bond may be critical to ligand utilization. Western ...
The ubiquitous presence of cell-surface sialic acid (SIA) has complicated efforts to identify specific transmembrane glycoproteins that function as bone fide entry receptors for influenza A virus (IAV) infection. The C-type lectin receptors (CLRs) DC-SIGN (CD209) and L-SIGN (CD209L) enhance ...
After separation by centrifugation (2,800 g for 10 minutes at 4°C) the pellet was resuspended in extraction buffer (1 M HCl, 0.02% β-βmercaptoethanol and protease inhibitor) and incubated at 4°C overnight. Pellet was extracted twice. The supernatants were pooled and treated with 10 ...
10 μg of resultant protein extract from each sample was resuspended in 1X NuPage LDS Sample Buffer (Invitrogen) prepared with 2.5% beta-mercaptoethanol and boiled at 95 ℃ for 5 min. Samples were loaded on a precast NuPage 10% Bis–Tris gel (Invitrogen) and run with 1X NuPage MOPS ...
In the co-immunoprecipitation assay, proteins were extracted from whole larvae of 6th-96 h using a binding buffer [10 μM Tris-HCl, pH 7.5; 2 μM β-mercaptoethanol; 1 μM EDTA (Ethylenediaminetetraacetic Acid), 10 mM PMSF (Phenylmethanesulfonyl fluoride)] and harvested by centrifugation at...
Bulk purified immune cells were incubated in DMEM with high-glucose supplemented with 10% FCS, 10 mM Hepes, 1 mM sodium pyruvate, non-essential amino acids, 80 μM 2-mercaptoethanol, 2 mM glutamine, 100 U ml−1 penicillin, 100 μg ml−1 streptomycin (all from Gibco...
Monocytic myeloid-derived suppressor cells (M-MDSCs) are significantly expanded in the blood of colorectal cancer (CRC) patients. However, their presence a
Subsequently, 1 mL IP buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA, 10 mM β-mercaptoethanol (β-ME)) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and Complete protease inhibitor cocktail (Roche) were added. After centrifugation at 20,800 x ...
(pH 8), 0.1 M NaCl, 1.0 mM β-mercaptoethanol, 5% glycerol, 0.5 mM imidazole Triton X-100 0.02%). Cells were sonicated (Sonifier Cell DisruptorB-30; Branson Sonic Power. Co., Danbury, CT) on ice using 12 bursts of 20 s at output level 5.5. After centrifugation at 4...
we derived the cell line ECN90 that is cultured at 37 °C in 5% CO2in Advanced DMEM/F12 medium (Thermo Fisher Scientific) supplemented with 2% bovine serum albumin fraction V (Roche), 6.7 ng/ml sodium selenite, 10 mM nicotinamide (Calbiochem), 50 μM β-mercaptoethanol (Sigma-Aldrich) an...