Figure 3.

Analysis of the in vitro translation products of human G-substrate cDNA and bacterially expressed recombinant human G-substrate. (A) The in vitro transcription–translation products were subjected to SDS/PAGE followed by autoradiography. Lane 1, pBluescript containing full-length human cDNA; lane 2, no template DNA; lane 3, pBluescript containing full-length human cDNA with heat treatment; lane 4, no template DNA with heat treatment. (B) Human G-substrate was expressed as a 6×His-tagged fusion protein in E. coli (BL21), purified, and analyzed by SDS/PAGE. Lane 1, crude extract; lane 2, heated crude extract; lane 3, after purification by chelating-Sepharose. The gel was stained with Coomassie brilliant blue. (C) Immunoblot of 6×His-tagged human G-substrate proteins with antibodies against rabbit G-substrate; lane 1, crude extract; lane 2, heated crude extract; lane 3, after purification by chelating-Sepharose; lane 4, purified rabbit G-substrate (10 ng).