X tremegene transfection reagent

HONE1, HK1, and NPC43 cells with low THY1 expression were used as recipient cells for THY1 restoration with stable expression of THY1, mediated by the pLVX expression vector [20 (link)]. The packaging vectors psPAX2 and pMD2.G were obtained from Addgene. The pLVX-EF1α-THY1-expressing vector was constructed. Lentivirus packaging was performed using 293T cells and X-tremeGENE transfection reagent (Millipore, Burlington, MA, USA, #6366236001). The cells were incubated for the production of lentivirus-containing medium for 2 days. Subsequently, the medium was harvested and stored at −80 °C until usage. For infection, NPC cells were trypsinized and then mixed with the lentivirus-containing medium at a ratio of 1:1 in a culture flask, with the addition of polybrene (Santa Cruz, Santa Cruz, CA, USA, #sc-134220). After 12 h, the medium was replaced with fresh, complete medium. Puromycin (Invitogen, Waltham, MA, USA, #ant-pr-1) was used for selection of successfully infected cells.

Jurkat cells were transfected with Ack1-specific siRNA (Dharmacon RNA Technologies, Lafayette, CO) or control siRNA by using X-tremeGENE transfection reagent (Sigma-Aldrich). Cells were allowed to culture for 48 hr and then harvested for immunoblotting experiments.

Plasmids encoding the layer mutant gp120 extended core (core_e) proteins, HIV-1_93TH057 gp120 core_e LM+HS and LM+HT, were transfected into GnT1⁻ cells using Xtremegene transfection reagent (Sigma-Aldrich) per the manufacturer’s instructions. Following 7 days of culture growth at 37°C and 8% CO₂, cells were pelleted by centrifugation and cell supernatant was filtered. Expressed gp120 was purified by passage over a 17b affinity column made by cross-linking MAb 17b to protein A (Pierce protein A IgG plus orientation kit; Thermo Fisher). gp120 was eluted with 0.1 M glycine (pH 3.0) into tubes containing 1/10 the final volume of 1 M Tris-HCl (pH 8.5) to raise the pH. The protein was then deglycosylated with 10 units/μg of Endo H_f (New England Biolabs) overnight at 37°C in a mixture of deglycosylation buffer, 50 mM sodium acetate (pH 6.0), and 350 mM sodium chloride. Endo H_f was removed by passage over an amylose resin column, and the protein was further purified by gel filtration chromatography on a Superdex 200 16/60 column (GE Healthcare, Piscataway, NJ) equilibrated with 5 mM Tris-HCl (pH 7.2) and 150 mM sodium chloride. The protein was concentrated to approximately 5 mg/ml for use in crystallization trials.