GALC in the ER has substantial enzyme activity. A, Purified ER fractions from total cell lysates of transfected HEK-293T cells showed that the 80 kDa GALC precursor form is present in the ER. GRP78 was used as an ER loading control. B, GALC activity assay performed with ER fractions reveals that the precursor protein possesses some enzymatic activity. Error bars indicate SD of triplicates. *p < 0.05 (Student's t test). **p < 0.01 (Student's t test). ***p < 0.001 (Student's t test). C, Purified lysosomal fractions contain mainly the processed GALCs. D, The same lysosomal fractions were also used for determination of GALC activities. Error bars indicate SD of duplicates. E, The GALC activities from ER (red bar) and lysosome (blue bar) were combined to show the relative contributions to actual cellular activity. F, Percentage of the activities from each subcellular organelle shows that the GALC in ER contributes significantly to the total activities in the mutants (G286D, T529M, and Y567S), but not in WT.