Figure 9.
Stabilization of membrane domains by antibody cross-linking. BHK cells expressed PLAP after transient transfection or VSV-G introduced by VSV infection. PLAP (left) and VSV-G (right) were patched using an mAb against PLAP and polyclonal antibodies against VSV-G, respectively, followed by the respective secondary antibodies. Cells were subsequently lysed in 2% Triton X-100 at 4° or 30°C. Fractions of an Optiprep™–sucrose flotation step gradient were analyzed by Western blot using anti-PLAP and anti–VSV-G antibodies. Antibody-induced patching (+Ab) significantly increases the amount of PLAP associated to a Triton X-100–insoluble membrane fraction that floats to the interphase of 20% Optiprep™/10% sucrose and 0% Optiprep™/ 10% sucrose, whereas VSV-G remains in high density fraction irrespective whether it was cross-linked or not.