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. 1997 Oct 20;139(2):485–495. doi: 10.1083/jcb.139.2.485

Figure 2.

Figure 2

Immunoblot analysis of PS-2 proteins. (A and B) PS-2 NH2-terminal, loop, and COOH-terminal sequences were expressed as GST fusion proteins and separated by SDS-PAGE on 8.5% polyacrylamide gels. The separated proteins were either stained with Coomassie blue (A) or transferred onto nitrocellulose filters and immunoblotted with the PS-2 NH2-terminal antibody (B). Lanes 1–4 are whole cell lysates of bacteria induced for expression of GST fusion proteins by addition of IPTG, and lanes 5–8 are the corresponding purified GST fusion proteins. The GST fusion proteins that were expressed are as follows: Lanes 1 and 5, GST alone; lanes 2 and 6, GST– NH2-terminal fusion protein; lanes 3 and 7, GST–loop fusion protein; and lanes 4 and 8, GST–COOH-terminal fusion protein. The arrowheads indicate the position of full-length GST fusion proteins. (C–F) Protein lysates of transfected HeLa cells separated by SDS-PAGE on 8.5% gels and immunoblotted with antibodies that recognize the transfected proteins. (C) Immunoblot with the anti–PS-2–specific antibody. Lanes 1–7 are lysates of cells transfected with the following constructs: Lanes 1 and 3, mock-transfected cells; lane 2, pPS-2; lane 4, pPS-2+Myc; lane 5, pPS-2(268aa)+Myc; lane 6, pPS-2(222aa)+Myc; and lane 7, pPS-2(166aa)+Myc. (D) Immunoblot of corresponding lysates shown in C with the anti-myc antibody. Lysates in lanes 1–5 in D correspond to lysates in lanes 3–7 in C. (E) Immunoblot with the anti–GFP-specific antibody. Lanes 1–4 are protein lysates of cells transfected with the following constructs: Lane 1, mock-transfected cells (control); lane 2, pGFP; lane 3, pGFP–PS-2; and lane 4, pGFP–PS-2(N141I). (F) Immunoblot of nonfusion PS-2 proteins with the NH2-terminal PS-2 antibody. Lanes 1–3 are 100 μg of protein lysates of cells transfected with equivalent amounts of the following nonfusion PS-2 constructs: Lane 1, mock-transfected; lane 2, pPS-2 (wild-type); and lane 3, pPS-2(Asn141Ile) mutant. The positions of protein molecular weight markers are indicated. Full-length PS-2 containing polypeptide bands seen only in transfected cell lysates are marked with large arrowheads. The small arrowheads indicate the positions of larger PS-2 complexes.