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. 2016 May 30;17(8):908–922. doi: 10.1111/tra.12404

Figure 2.

TRA-12404-FIG-0002-b

Proteolytic cleavage of WT and mutant GALC. A) Protein lysates from either untransfected HEK293T cells or from cells transfected with WT GALC were subjected to immunoprecipitation with monoclonal antibodies against full‐length human GALC (mAb1 and mAb2) or the FLAG epitope tag. GALC was detected using western blot with a polyclonal rabbit GALC antibody, cleavage product at ∼30‐kDa is marked with an asterisk. B) Conditioned media (CM) from HEK293T cells transfected with WT GALC contains full‐length GALC (lane 1). GALC‐containing CM (+) and CM from untransfected cells (−) was harvested after 72 h and applied to untransfected HEK293T cells for 48 h. Treated cells were subjected to immunoprecipitation using the GALC monoclonal antibody mAb2 and detected as in A. C) HEK293T cells were transiently transfected with WT GALC and Krabbe mutations R380W, E215K, R515H and T513M and immunoprecipitated using the mAb2 antibody and detected as in A.