Protein Immunoprecipitation and Western Blot

Protein immunoprecipitation and Western blot analysis were performed using standard protocols, as previously described [21 (link),24 (link)]. In brief, 293-VnR cells transfected with ADAM12-GFP or with basigin were extracted in RIBA buffer for 20 min, containing inhibitors as described [20 (link),23 (link)]. The extracts were incubated with antibodies for 2 h at 4 °C with gentle agitation. Protein G-SepharoseTM 4 Fast Flow beads (GE Healthcare, Chicago, IL, USA) were added for an additional 1 h at 4 °C. Beads were gently washed three times in RIPA buffer (20 mM Tris-HCl (pH 7.5) 150 mM NaCl, 1 mM Na2EDTA, 1% Triton X-100). Bound proteins were eluted in 2× sample buffer, followed by Western blot analysis. Cell surface proteins were biotinylated using non-cleavable EZ-Link Sulfo-NHS-LC-Biotin, pulled down with streptavidin–agarose, and analyzed by Western blotting as previously described [44 (link)].