Immunoprecipitation and crosslinking of Fpn demonstrate Fpn is a multimer. (A) HEK293T cells were transfected with pFpn-FLAG and pFpn–c-myc. Twenty-four hours after transfection cells were solubilized in lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.2, 50 mM EDTA, 1% Triton X-100 with 1X protease inhibitor cocktail) and Fpn-FLAG immunoprecipitated as described in “Materials and methods.” The input, flow-through (FT), and eluate were examined for the presence of Fpn-FLAG and Fpn–c-myc by SDS-PAGE and Western blot analysis. The volumes of the input and flow-through analyzed were twice that of the eluate. (B) HEK293T cells were transfected with pFpn-GFP and pFpn-FLAG. Twenty-four hours after transfection cells were placed at 0°C and incubated with 1.5 mM crosslinking reagent EGS for 60 minutes. The crosslinking reagent was quenched by addition of cell growth medium, cells were solubilized in lysis buffer as in panel A, and the lysates were incubated in the presence or absence of the cleaving reagent, 1.0 M hydroxylamine-HCl, for 60 minutes at room temperature. The extracts were examined for the presence of crosslinked high-molecular-mass Fpn-GFP and Fpn-FLAG by 4% SDS-PAGE and Western blot analysis (left panel) or 8% SDS/PAGE and Western blot analysis after cleavage of the crosslinking.