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Figure 1.
Figure 1. CXCL14 expression is regulated by the 26 S
proteasome and is a substrate for ubiquitylation. (a) Primary
prostate epithelial cells (PrEC) and the prostate cancer cell
line LNCaP were transduced for three days with CXCL14 expressing
virus (pAd-CXCL14) or vector-only virus (pAd). The conditioned
media were analyzed by Western blotting for CXCL14 expression.
(b) Total RNA was isolated from the transduced cells, converted
to cDNA and reverse transcriptase PCR performed using primers
designed to detect CXCL14 or GAPDH. PCR products were separated
by agarose gel electrophoresis and detected by SYBR green
staining. Neg, denotes the no DNA control. (c) LNCaP cells were
transduced with CXCL14 expressing adenovirus (pAd-CXCL14) for 48
h. Inhibitory doses of chloroquine (10 μM) and MG-132 (10 μM)
were added for an additional 24 h. The conditioned media were
analyzed by Western blotting for CXCL14 expression.
Un-transduced LNCaP cells were included as a control treated
with vehicle only (dimethyl sulfoxide, DMSO). (d) LNCaP cells
expressing CXCL14 were treated for 24 h with increasing
concentrations of lactacystin. The conditioned media were
analyzed by Western blot analysis for CXCL14 expression. (e)
LNCaP cells were transduced with CXCL14 expressing adenovirus
(pAd-CXCL14) or vector alone. After 48 h post-transduction,
cells were treated with 10 μM MG-132 or DMSO. Cells were
harvested, lysed, and immunoprecipitated using the anti-CXCL14
antibody. Immune complexes were subjected to Western blot
analysis and probed with a mouse anti-ubiquitin or the rabbit
anti-CXCL14 antibody. A high molecular weight complex was
detected which is indicative of polyubiquitylated proteins. (f)
Cells were transduced with vector (pAd) or CXCL14 expressing
(pAd-CXCL14) adenovirus. After 48 h post-transduction, cells
were treated with MG-132 or DMSO. The media were analyzed for
CXCL14 expression by Western blot analysis. Without pAd-CXCL14
transduction the protein was undetectable in all cell lines.
Primary cells utilized include: prostate epithelial cells, PrEC;
human urothelial cells, HUC; and kidney epithelial cells, KEC.
The immortalized PrEC lines include: HPV15E6, HPVE6 oncoprotein
immortalized; and HPV15E7, HPVE7 oncoprotein immortalized.
Cancer cell lines include: CWR22, DU145, LNCaP, LAPC4, PC-3,
PPC-1, 293 and HeLa.
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