Skip to main content
. 2014 Mar 10;289(17):11630–11641. doi: 10.1074/jbc.M114.555870

FIGURE 6.

FIGURE 6.

Characterization of domain swapped chimeric HPC4-sTsyn and HPC4-sβ4GalT1. A, primary aa sequence showing CBRT swapped HPC4-sTsyn (red) and HPC4-sβ4GalT1 (green) and schematic showing chimeric HPC4-sTsyn and HPC4-sβ4GalT1. B, pulldown experiment demonstrating significant loss of HPC4-sT-synΔCBRT binding to Cosmc and gain of HPC4-sβ4GalT1-CBRT binding to Cosmc. Input, unbound, and bound to Cosmc fractions from the pulldown experiment were analyzed by antibody against HPC4 for T-synthase/β4GalT1. Similarly, the amount of Cosmc used in the pulldown experiment was analyzed by monoclonal antibody against Cosmc (bottom panel). C, β4GalT1 activity was determined from whole cell lysates from Hi-5 cells expressing HPC4-sβ4GalT1 or HPC4-sT-syn or chimeric versions expressed individually or co-expressed with His-sCosmc (top panel). The data show averages of three independent experiments. Error bars, ± S.D. from the average. HPC4 containing either T-syn or β4GalT1 or their respective chimera were detected by Western blotting against HPC4 (bottom panel), and a representative Western blot from three is shown. D, similar to C, T-synthase activity and HPC4 containing proteins were determined. The data show averages of three independent experiments performed in duplicate. A representative Western blot from three independent analyses is shown. Error bars, ± S.D. from the average. WB, Western blot.