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. 2016 Dec 13;17(12):2089. doi: 10.3390/ijms17122089

Figure 2.

Figure 2

Figure 2

Enzyme activity and protein expression of rhα-GLA in the GLA-null cell lines. The CRISPR/Cas9-mediated GLA-null HEK-293T cell lines, i.e., clone 19 (A) and clone 30 (B) were incubated with 0.1 and 1 µM Replagal or Fabrazyme for 6 h. The GLA enzyme activities were measured based on the description of “Materials and Methods” 1 day after rhα-GLA treatment and the results were presented as mean ± SD (n ≥ 3). In addition, the rhα-GLA protein expression in cell lysates was determined by immunoblot probing of the monoclonal antibody against the human GLA. β-actin was used as loading control. Representative results out of three independent experiments are presented; (C) Time schedule determining the intracellular pharmacokinetics of rhα-GLA was illustrated. Briefly, the GLA-null cell lines were treated with 0.1 µM Replagal (D) or Fabrazyme (E) for 24 h. Following the treatment, the rhα-GLA was replaced with fresh medium and the intracellular rhα-GLA protein levels and the corresponding GLA enzyme activity were measured accordingly at the indicated time (from 6 h to 7 days). β-actin was used as loading control in the immunoblot assay. Representative immunoblot data are presented. In addition, the GLA enzyme activities are presented as mean ± SD (n ≥ 3).