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. 2002 Aug;22(16):5639–5649. doi: 10.1128/MCB.22.16.5639-5649.2002

FIG. 6.

FIG. 6.

Effects of mutation of arginines 252, 264, and 267 at the putative S1P cleavage sites on generation of the amino-terminal cleaved fragment of Luman. (A) Vero cells were transfected with plasmids expressing either FL-Lu-HA (Luman) or FL-Lu-HA(R252A) expressed from the CMV IE promoter or an HSV TK promoter. The CMV IE and HSV TK promoters containing blank plasmids, pcDNA3 and pTK3, were used as controls. Cells were cotransfected with pBB15, a reporter plasmid with coding sequences for CAT linked to the basal HSV LAT and pCMV-β-Gal. CAT activity is adjusted for transfection efficiency. (Inset) Western blot of lysates of cells transfected with the pTK constructs. Arrow indicates the location of bands representing Luman and Luman(R252A). The lower-molecular-weight band in all lanes is due to nonspecific binding of the anti-Luman antibody. (B) Lysates of cells expressing LuFL-Lu-HA(R252A), either left untreated (Lu), treated with brefeldin A (Lu + bref), or cotransfected with either pS1P-KDEL or pS1P-KDAS, were separated on SDS-10% polyacrylamide gels and probed with anti Luman serum. (C) Cells transfected with pBB15 and (from left to right) either pcDNA, pcFL-Lu-HA (Luman), pcFL-Lu-HA(R264G), pTK3, pTKFL-Lu-HA (Luman), pTKFL-Lu-HA(R264G), or pTKFL-Lu-HA(R267G) were assayed for CAT activity. (Inset) Western blot of lysates of cells transfected with the pTK constructs. (D) Cells transfected with plasmids expressing Luman or its mutants (R252A, R264G, or R267G), either alone or in conjunction with plasmids expressing either S1PKDEL or S1PKDAS, were analyzed by immunoblotting. Cells expressing Luman or mutant proteins alone were either left untreated (lanes 1, 5, 9, and 13) or treated with brefeldin A (lanes 2, 6, 11, and 14).