Cross-linking of Pg 1 to 1-LN cell membrane proteins.
1-LN cell monolayers grown in 150-cm2 culture flasks were gently scraped and suspended in 1 mL serum-free RPMI 1640. After incubation with Pg 1 (1 μM), cell lysates were prepared and the cross-linked proteins were purified by immunoaffinity on anti-Pg IgG-Sepharose as described in “Materials and methods.” Aliquots (30 μL) were resolved by SDS/10%-PAGE under reducing conditions, and the proteins were transferred to nitrocellulose membranes and incubated with specific anti-Pg and anti-TF IgG. Lane 1, reaction of cross-linked proteins from 1-LN cells incubated with Pg 1 and goat anti-human Pg (Fab')2 fragments. Lane 2, reaction of solubilized 1-LN cell membranes (30 μL) with goat anti-human Pg F(ab')2fragments. Lane 3, Pg 1 (1 μg) reacted with goat anti-human Pg F(ab')2 fragments. Lane 4, Pg 1 (1 μg) reacted with a rabbit anti-goat alkaline phosphatase-conjugated F(ab')2fragments. Lane 5, reaction of cross-linked proteins of 1-LN cells incubated with a goat anti-human TF F(ab')2 fragments. Lane 6, reaction of solubilized 1-LN cell membranes (30 μL) with a goat anti-human TF F(ab')2 fragments. Lane 7, isolated 1-LN TF (1 μg) reacted with a goat anti-human TF F(ab')2fragments. Lane 8, reaction of isolated 1-LN TF (1 μg) with a rabbit anti-goat alkaline phosphatase-conjugated F(ab')2fragments.
