Figure 1.

A. LNCaP-PLNX7 cells were transfected with a plasmid, PL-N-NKX3.1, allowing the expression of a chimeric NKX3.1 protein fused to the ProLabel tag at its C-terminal end. Upon transcription and translation, the PL-N-NKX3.1 fusion protein was phosphorylated and subjected to degradation through the ubiquitin-proteasome pathway. As the enzyme donor, the ProLabel tag in PL-N-NKX3.1 fusion protein can associate with enzyme acceptor (EA) to form an active enzyme that cleaves the chemiluminescent substrate; the resulting signal can be detected with a standard luminometer. Therefore, PL-N-NKX3.1 protein levels could be easily determined by cell-based high-throughput screening assays. B. PL-N-NKX3.1 was transiently transfected into 293T cells and the fusion protein was stained with NKX3.1 antiserum. C. Immunoblots from LNCaP cells transiently transfected with PL-N-NKX3.1 and 48 hr after transfection, cells were treated with cycloheximide for the indicated times. D. Immunoblot of extracts from a derivative LNCaP cell line expressing PL-N-NKX3.1 (clone #7, or PLNKX7). Cells were treated with cycloheximide and harvested at the indicated times for detection with NKX3.1 antiserum. E. Quantification of the immunoblot in D by densitometric scanning. F. LNCaP-PLNKX7 cells were transfected with NKX3.1 siRNA by reverse transfection. Immunoblotting was performed with NKX3.1 antiserum for both PL-NKX3.1 and endogenous NKX3.1. G. Sensitivity of the LNCaP-PLNKX7 detection assay was demonstrated in 96-well plates at an LNCaP cell density of 7000 cells/well. Cells were lysed directly in 96 well plate 4 days after transfection and chemiluminescence was determined.