Western_blot_LBP_D283G_in_HEK293T_overexpression

Western blot of transiently transfected HEK 293T cells with plasmids encoding ancestral or derived (D283G) LBP cDNAs, including the previously reported P333L hypomorphic variant, for comparison purposes. Transient transfection was carried out in HEK293T cells transfected with 1 µg of plasmid DNA in the presence of X-tremeGene9 DNA transfection reagent (#6365809001, Merck), according to the manufacturer’s instructions. Transfected cells were cultured at 37°C, under an atmosphere containing 5% CO2, in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum. After 48 h, supernatants and whole-cell lysates were collected for subsequent experiments. Whole-cell protein lysates were extracted in modified radioimmunoprecipitation assay buffer supplemented with protease inhibitors (#5892970001, Merck) and phosphatase inhibitor cocktail (#4906837001, Merck), 0.1 mM dithiothreitol (DTT; Life Technologies), and 1 mM PMSF (#10837091001, Merck). Protein extracts and supernatants were resolved by electrophoresis in Criterion TGX 10% precast gels (Bio-Rad), with the resulting bands transferred onto PVDF membranes (#1704157, Bio-Rad) with the Transblot turbo system (Bio-Rad). Membranes were probed by incubation for 1 hour at room temperature with antibodies against LBP (#AF870-SP, R&D Systems, 1:2,000), DDK (#A8592, Merck, 1:10,000) and GAPDH (#sc-47724, Santa Cruz Biotechnology, 1:5,000). Proteins were detected by chemiluminescence with Clarity Western ECL substrate (#1705061, Bio-Rad) reagents. Importantly: Figures are flipped and rotated for the ease of imaging capture.