LRP-1 regulates cell-surface TNFR1. (A) PEA-10, MEF-2, and B-41 cells were grown for 18 hours in SFM. Cell-surface proteins then were biotin-labeled. The biotin-labeled proteins were purified by streptavidin-Sepharose affinity chromatography and then subjected to immunoblot analysis for TNFR1 (TNFR1 surface). Total cell extracts were also subjected to immunoblot analysis for TNFR1 (TNFR1 total) and for total ERK/MAP kinase as a control for protein load (vertical lines have been inserted to represent a repositioned gel lane). (B) PEA-10 and MEF-2 cells were incubated with biotin-labeled human TNF-α, followed by fluorescein isothiocyanate-labeled streptavidin. The cells then were subjected to FACS analysis. Control preparations were incubated with biotin-labeled soybean trypsin inhibitor (SBTI), which does not bind to cell-surface proteins. (C) ¹²⁵I-labeled TNF-α was incubated with PEA-10 or MEF-2 cells for 4 hours at 4°C. The cells were washed and lysed in 0.1 M NaOH, 1% SDS. Radioactivity associated with the cell lysates was determined using a γ-counter. Error bars represent SD. (D) Cell-surface proteins from CL-16 cells and shLRP-1 cells were labeled with biotin and purified by streptavidin-Sepharose chromatography. The purified proteins were subjected to immunoblot analysis for TNFR1 (TNFR1 surface) or LRP-1. Total cell extracts also were subjected to immunoblot analysis for TNFR1 or tubulin as a measure of protein load.