Detection of direct interaction between partially purified FLAG-tagged CerS1 and recombinant His-p17/PERMIT proteins in vitro

A549 cells transfected with the hCerS1WT-FLAG, hCerS1Δ60–76-FLAG, or empty vector were lysed by freeze-thawing in a lysis buffer containing 50 mM tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, and 0.5% NP-40 with a protease inhibitor cocktail (1:500). Cell lysates (400 μg of protein) were incubated with 150 μl of magnetic beads conjugated with antibody against FLAG (Sigma-Aldrich) for 18 hours at 4°C with agitation. After extensive washes, CerS1-FLAG containing beads were incubated with 30 μg of His-tagged p17/PERMITWT or His-tagged p17/PERMITR28–30A recombinant purified proteins in 150 μl of binding buffer containing 50 mM tris (pH 7.5), 137 mM NaCl, 1 mM MgCl2, 2.7 mM KCl, 15 mM NaF, and 0.5 mM NaV3O4 overnight at 4°C with agitation. This was followed by several washes after proteins bound to the beads were eluted using 1× SDS loading buffer without βME at 95°C for 3 min. The resulting eluate was loaded on the Criterion TGX Stain-Free Precast Gel (Bio-Rad), followed by Western blot analysis with anti-FLAG (Sigma-Aldrich) or anti-His (Invitrogen) antibodies.

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