Figure 2. Transient expression of arylsulfatase-related genes. A and B: Endogenous PDI (ER marker, A) and cathepsin D (lysosome marker, B) were immunostained. ARPE-19 cells were transfected with 6 μg of the expression vector ARSI-FLAG. Scale bars, 10 μm. C: The structures of FLAG-tagged protein constructs were depicted. The colored box at the N-terminal represents a signal sequence. The active site of ARSI, Cys (C), was replaced by Ser (S) in a mutant derivative. The size of each secreted mature protein was represented. D: The expression products were analyzed with western blot. ARPE-19 cells were transfected with 1 μg SUMF1-FLAG, 5 μg ARSA-FLAG, or 8 μg ARSI derivatives, and cultured in serum-containing media for 22 h and then in serum-free media for 9 h. Loaded were 20% of the total sample for insoluble fraction, 10% for cell extract, and 25% for medium. The lane number corresponds to the construct number of the FLAG-tagged protein described in C.