Fig. 2

a Expression and purification of N-terminal TTC fusion proteins in prokaryotes. Each cDNA coding TTC or TTC-Bcl2 was introduced into a prokaryotic expression vector with an N-terminal 6 × His tag sequence (His- His- His- His- His- His). CBB staining was used for detection of both crude and purified TTC. TTC-Bcl2 fusion proteins were further detected with anti-Bcl-2 antibody (arrowhead). b Cell surface binding of fusion proteins. Binding of approximately 40 nM (as estimated from SDS-PAGE) TTC or TTC-Bcl2 to NGF-differentiated PC12 cells immuno-detected with an anti-His tag antibody. Purified TTC bound to the cell surface of neuronal-differentiated PC12 cells whereas TTC-Bcl2 fusion protein did not. The results suggest that binding to neuronal cells could require a free TTC C-terminus. Scale bar = 25 μm. c Expression and purification of N-terminal TTC fusion proteins in prokaryotes. Bcl2-TTC and Bcl2-TTH fusion proteins, as well as Bcl-2 alone, tagged with 6 × His tag sequence at the N-terminus were expressed in E. coli. Fusion proteins detected with an anti-Bcl-2 antibody (arrowhead). For Bcl2-TTC and Bcl2-TTH, preparations of purified fusion proteins contained smaller-sized fragments (30 kDa) that likely represent C-terminal truncated proteins. d Cell surface binding of fusion proteins. Binding of approximately 40 nM (as estimated from SDS-PAGE) of fusion proteins to NGF-differentiated PC12 cells immuno-detected with an anti-His tag antibody. Purified Bcl2-TTC bound to the cell surface of neuronal-differentiated PC12 cells, whereas Bcl-2 and Bcl2-TTH fusion proteins did not. Scale bar = 25 μm. e Cell viability assays. PC12 cells were differentiated by NGF and then exposed to each of the recombinant proteins for 1 h. After exposure, serum and NGF were withdrawn. PC12 cell survival was evaluated 48 h after exposure. Compared to control only, Bcl2-TTC significantly increased PC12 cell survival following serum and NGF withdrawal (*: p < 0.05). Values are means ± s.e. (n = 8)