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. 2008 Oct 17;283(42):28497–28505. doi: 10.1074/jbc.M805092200

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Copyright © 2008, The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 2. — The PA domain of GRAIL directly binds tetraspanins. A, left, 293T cells were transfected with expression constructs for V5-tagged GRAIL and 3×HA-tagged CD81 along with empty control vectors. Cell lysates were immunoprecipitated (IP) with anti-HA and immunoblotted (IB) with anti-V5 and anti-HA. Right, 293T cells were transfected with various V5-tagged expression constructs for wild-type GRAIL and GRAIL mutants along with 3×HA-tagged CD81. Cell lysates were immunoprecipitated with anti-HA and immunoblotted with anti-V5 or anti-HA. B, 293T cells were transfected with either 3×HA vector and V5-tagged GRAIL, 3×HA-tagged CD81 and V5 vector only, or 3×HA-tagged CD81 and V5-tagged GRAIL. Cells were lysed in 1% Brij96, lysates were divided in half, and one-half was cross-linked with DSP on ice for 2 h. Free DSP was neutralized with Tris, and 0.1% SDS was added to each sample. Cell lysates were then immunoprecipitated with anti-HA and immunoblotted with anti-GRAIL or anti-HA.