Since the overexpression of during OPH in tumors compared to normal prostate tissue is the most ideal situ ation for OPH targeted prodrugs, we have limited the re mainder of this study to the non tumorigenic RWPE 1 and tumorigenic LNCaP cell lines. Esterase activity profiles with n PAGE electroblotting In order to further validate the nanospray LC MS MS results we next tested the possibility that, esterase activity could be maintained after n PAGE electroblotting, immunostaining could be used to confirm the pres ence of OPH protein in the n PAGE esterase activity bands, nanospray LC MS MS could be performed on the electroblotted esterase bands. RWPE 1 and LNCaP ly sates were separated on 6% n PAGE gels and the proteins transferred to a nitrocellulose membrane by electroblot ting and the esterase bands visualized by activity staining of the membrane with S ANAA substrate.

As shown in Figure 5A, this methodology resulted in the appearance of two additional sharp bands in the 220 240 kDa native protein marker region of the blot. A parallel blot was probed with anti OPH antibody to confirm the proteomic identification of OPH within the activity bands. The ester ase activity was quantified using densitometry analysis and the LNCaP activity bands showed about a 50% higher esterase activity compared to the respective RWPE 1 activity bands. Densitometry of the anti OPH immunoblot revealed relative intensity pat terns that paralleled that seen for the activity bands. The four OPH activity bands were excised separately and each band analyzed by LC MS MS.

As detailed in Table 2, OPH was identified within the most active activity bands but could not be consistently identified within the least active band. We also noted that the es terase activity profiles with n PAGE electroblotting had a lower level of background staining than similarly stained native gels. OPH accounts for the all the esterase staining observed with the S ANAA substrate We next investigated whether the apparent esterase ac tivity with the S ANAA substrate observed in the 198 and 216 kDa bands was due completely to OPH. Native PAGE gels run with LNCaP or RWPE 1 lysates were pre incubated with 50 uM diisopropyl fluorophosphate, a known irreversible inhibitor of serine esterases proteases and of OPH, before activity staining with S ANAA substrate. The 198 kDa and 216 kDa esterase bands showed no visible activity after pre incubation with DFP, indicating that the esterase selleck chemicals activity observed was completely due to a serine hydrolase activity. We further confirmed this finding by pre clearing the cell lysates with anti OPH antibody prior to n PAGE esterase activity pro filing.