Fig. 4: Biochemical characterization of HPCA as a putative MAG protein ligand.

A A representative Coomassie gel showing the purity of recombinant N-terminal 6x-His tagged mouse HPCA from E. coli using affinity chromatography. (B) The UV-dependent preferential binding of the PG-DA probe to recombinantly purified mouse HPCA. In this experiment, the HPCA and lipid probe (PG-DA or PA-DA) concentrations were 10 μM and 10 μM respectively. (C) The dose-dependent binding of the PG-DA probe (0 – 250 μM) or the PA-DA probe (0 – 250 μM) to recombinantly purified mouse HPCA (10 μM). This experiment also shows that at comparable probe concentrations, relative to the PA-DA probe, the PG-DA probe binds much better to HPCA. (D) The effect of excess calcium (CaCl₂, 100 μM), or EDTA (100 μM) on the binding of the PG-DA probe (10 μM) to recombinantly purified mouse HPCA (10 μM). (E) The effect of incubating different 1-MAG variants (200 μM) on the binding of the PG-DA probe (10 μM) to recombinantly purified mouse HPCA (10 μM). (F) The effect of incubating increasing concentrations of 1-PG (0 – 250 μM) or PA (0 – 250 μM) on the binding of the PG-DA probe (10 μM) to recombinantly purified mouse HPCA (10 μM). For all representative gels shown in (B–F), the top panel shows the in-gel fluorescence, while the bottom panel is the Coomassie staining as the loading control for that respective gel. For all representative gels shown in (B–F), the red asterisk denotes the minor form of HPCA at 25-kDa in both gels. For all representative gels shown in (B–F), the Coomassie gel requires over-exposure to visualize this minor band of HPCA. For all gels shown in (B–F), the UV exposure time was 6 mins. All experiments shown in this figure (A–F), were done at least three times with reproducible results each time.