The physical association between RECK and LIFR or gp130 was analyzed by two different but complementary methods: IP/IB as previously described [27 (link), 28 (link)] and double immunofluorescence. For IP, equal amounts of whole cell extracts were incubated with anti-RECK antibodies immobilized on agarose beads under slow rotation. After washing in a buffer containing 50 mM Tris–Cl, 150 mM NaCl, and 0.1% Nonidet P-40 three times, the bound proteins were eluted by boiling in SDS sample buffer, run on SDS–PAGE and IB with LIFR or gp130 antibodies. The antibodies against RECK, LIFR, and gp130 are described above. For immunofluorescence, cells seeded at a density of 2,000 cells on a glass slide were fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton-X100 (30 min at 4°C), washed and then incubated with anti-RECK and anti-LIFR or anti-gp130 antibodies (1 hr at RT) followed by secondary antibodies conjugated with Alexa flour 488 or Alexa flour 594 antibodies (Abcam) and nuclear localizing DAPI (4′,6-diamidino-2-phenylindole dihydrochloride, #D1306, ThermoFisherScientific) at RT for 1 hr. Omission of primary antibodies failed to provide specific signals and served as controls. Images were captured by confocal microscopy (Leica Microsystems, Wetzlar, Germany). Red and green signals were colocalized along with DAPI (blue) in merged images.
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