Immunoprecipitations using perilipin and vimentin antibodies and briefly AIM-stimulated preadipocytes.

Mabs Peri112.17, Vim3B4 and control VE-cadherin were used. Immunoprecipitations (IPs) were analyzed by SDS-PAGE and CB staining (a) and corresponding WB using pab Peri-hNT (b). Lane 1: control unspecific binding of cell lysate to beads, i.e. magnetic beads after incubation with lysate and PBS washing; lane 2: bound lysate material of perilipin antibody beads; lane 3: control unbound supernatant of perilipin antibody beads; lane 4: bound lysate material of vimentin antibody beads; lane 5: control unbound supernatant of vimentin antibody beads; lane 6: bound lysate material of VE antibody beads; lane 7: control unbound supernatant of VE antibody beads; lane 8: control magnetic beads after incubation with crosslinked lysate; lane 9: bound crosslinked lysate material of perilipin antibody beads; lane 10: bound crosslinked lysate material of vimentin antibody beads; lane 11: bound crosslinked lysate material of VE antibody beads. Positions of molecular weight marker proteins are given on the left margin. Of importance, perilipin and notably vimentin antibodies precipitate and co-precipitate perilipin (lanes 2,4; perilipin position at 65 kD is marked at left margin by an arrow) in contrast to the negative control antibody (lane 6). In addition, similar positive reactions are obtained with crosslinked lysate by detecting crosslinked perilipin (lanes 9,10) whereas the control antibody shows no reaction with crosslinked material at all (lane 11).