Exposure cassette

Recombinant His6-tagged, solBGs (WT or AA) was incubated with ¹²⁵I-HGF or ¹²⁵I-TGFβ2 in PBS/Triton (0.05 %) for 3 h on a rocking platform. Protein complexes were incubated with Talon beads 30 μL/1 h, followed by three washes, before crosslinking with disuccinimidyl suberate (DSS, 60 μg/mL final concentration), for 15 min. The crosslinking was stopped with 10 mM Tris-HCl pH 7.4, containing 1 mM EDTA. Beads were washed and bound radioactivity was counted in a Cobra II auto-gamma (Packard). Then, the immunoprecipitated complexes were separated in SDS-PAGE and revealed by phosphor screening. For dot-blots, WT- and AA-solBGs were immobilized on PVDF membranes using a dot-blot chamber for 15 min, albumin was used as control. Vacuum was applied to aspirate the solBG preparations and each well was washed three times with PBS, blocked with 5 % milk in TBS-T for 1 h, followed by three washes with TBS-T. The labeled factors, ¹²⁵I-HGF (40,000 cpms) or ¹²⁵I-TGFβ2 (20,000 cpms) were incubated for 6 h at 4 °C on a rocking platform, then washed six times with TBS-T. Membranes were exposed on appropriate Phosphor screens (Exposure Cassette, Molecular Dynamics) for 2 days and revealed in a Typhoon FLA 7000 counter (General Electric).