Fig. 7.

Labeling of CD14 with 17ODYA is confined to its GPI anchor. (A) Scheme of experimental procedure. CD14 wild type (WT), CD14-VSVG, or CD14-VSVGmut were expressed in HEK293 cells. Cells were labeled with 50 μm 17ODYA or exposed to 0.05% DMSO carrier for 4 h, lysed, and cell lysates were subjected to click chemistry reaction with biotin-azide. Biotin-tagged proteins were captured on streptavidin-coupled beads, and, after elution, separated by SDS-PAGE. A parallel set of samples was subjected to delipidation after the click chemistry reaction (DeLip). (B) The presence of CD14 or flotillin-2 in eluates from streptavidin beads and in lysates (10% of total cell lysate). As CD14 WT did not contain the HA tag, all CD14 variants were detected with anti-CD14 antibody. Molecular weight markers are shown on the right. (C) Structure of CD14 constructs. TM, CP, transmembrane and cytoplasmic fragments of VSVG, respectively. HA, HA tag. Results of one representative experiment of three are shown.