FIG 8.

The relative contributions of K122, E123, and S124 R group properties to Asp14 PDI binding efficiency. (A and B) HeLa cells were transfected to coexpress Flag-PDI and GFP, GFP-Asp14, or GFP-Asp14 bearing the indicated amino acid substitution. Input lysates were subjected to Western blotting with GFP and Flag antibodies to confirm protein expression and with GAPDH antibody to confirm that the input lysates contained equivalent amounts of protein. Whole-cell lysates were incubated with Flag antibody-conjugated agarose beads to immunoprecipitate Flag-PDI and its interacting proteins. The resulting Western blots were probed with Flag antibody to confirm that Flag-PDI was recovered and GFP antibody to determine if Flag-PDI coimmunoprecipitated GFP or GFP-tagged Asp14 protein. (C) The efficiency with which Flag-PDI coprecipitated GFP or GFP-Asp14 fusion proteins was determined as described above. The mean normalized coprecipitation efficiency ± SD from three to six replicates per condition was determined. Statistically significant values are indicated. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Asterisks above columns indicate statistically significant differences relative to the results for GFP-Asp14, while asterisks over horizontal brackets denote statistically significant differences between the indicated column values.