FIGURE 5.

Competition between NAGS and mouse variable segment (mVS) for co-IP with CPSI. Recombinant mVS was overexpressed and purified from E. coli. (A) Amino acid sequences of recombinant mouse variable segment (mVS), reversed (revVS) and shuffled (shVS) variable segments. Amino acids from the C-terminal half of mVS (bold and underlined typeface) were interdigitated with amino acids from the N-terminal half to create shVS. (B) Purification of mVS, rVS and shVS. Lanes 1, 6 and 11—flow trough. Lanes 2, 7 and 12—wash with 50 mM imidazole. Lanes 3, 8 and 13—elution with 125 mM imidazole. Lanes 4, 9, and 14—elution with 250 mM imidazole. Lanes 5, 10 and 15—elution with 500 mM imidazole. (C) Anti-CPSI antibodies were used for immunoprecipitation of mitochondrial proteins. The precipitated proteins were probed with anti-mNAGS antibodies. Precipitation with non-specific antibodies (Neg), revVS (IP CPSI+revVS) and shVS (IP CPSI+shVS) were negative controls. Total liver proteins (Liver) were positive control.