FIGURE 9.

Effects of GEC1 and its analogues on FLAG-hKOPR expression. A, amino acid sequence alignment of GEC1, GABARAP, and GATE16 showing the highly conserved nature of the 12 GEC1 residues involved in interaction with hKOPR. B, GEC1 up-regulated FLAG-hKOPR to a higher extent than GABARAP, GATE16, and GEC1-(38–117). Ten μg of vector, GEC1, GEC1-(38–117), GABARAP, or GATE16 cDNA was transfected, and receptor binding assays were conducted 40 h later. Results are expressed as mean ± S.E. of three experiments. ^*, p < 0.05, and ^**, p < 0.01 compared with GEC1 group using one-way ANOVA followed by Tukey's post hoc test. C, immunoblotting of GABARAP family proteins and truncated GEC1. Immunoblotting of proteins at 40 h after transfection was conducted. Two hundred thousand cells in Laemmli sample buffer (per lane) were loaded and resolved by 15% Tricine/SDS-PAGE. Proteins were detected by immunoblotting with polyclonal anti-GEC1 antibody, which cross-reacts with GABARAP and GATE16 in immunoblotting (14).