pT120 β-catenin blocks generation of nuclear ABC and accumulates in trans Golgi network.

<p>(A) C4-2 and C4-2/PKD1 cells were fractionated into fractions enriched for cytoplasmic, nuclear or membrane proteins and analyzed by Western blot with antibodies as noted. E-cadherin, lamin A/C and b-actin were used as plasma membrane, nuclear and cytosol markers, respectively. The * indicates a leftover band from previous blotting. The densitometric data of each β-catenin isoform in a cell was the average of three assays and was expressed as percentage. (B) Overexpression of PKD1 prevents Wnt-induced β-catenin accumulation. C4-2 and C4-2/PKD1 cells were cultured with recombinant Wnt3a for indicated times. The relative amount of β-catenin at each time point was expressed as a ratio to the amount of β-catenin at starting time 0 (data was from a single assay). (C) Immunofluorescence staining of C4-2/PKD1 cells with pT120 and TGN p230 antibodies The TGN and pT120 colocalization are indicated by white arrows.</p

Analysis of pT120 antibody staining in prostate cancer.

<p>Analysis of pT120 antibody staining in prostate cancer.</p

Characterization of pT120 antibody.

<p>(A) NIH 3T3 lysates that were transfected with either HA-tagged wild type, or T102I/T112R/T120I triple mutant, or T120I mutant β-catenin and blotted by pT120 antibody. (B) Cell lysates from C4-2 (lanes 1 and 3) and C4-2/PKD1 (lanes 2 and 4 to 7) cells were blotted with either β-catenin conventional antibody H102 (lanes 1 and 2) or pT120 antibody (lanes 3–7). Competition assay was performed in C4-2/PKD1 cell lysate in the presence of 20 nM of antigenic phospho-peptide (lane 6) or the nonphospho-peptide (lane 7). (C) Inhibition of PKD1 activity decreases T120 phosphorylation. LNCaP cells were transiently transfected with two shRNA targeting PKD1 or a control shRNA vector. Cell lysates were blotted with antibodies indicated. The relative protein levels quantified by densitometry are expressed as fraction of the control group on the average of two assays.</p

pT120 β-catenin is significantly decreased in TGN of prostate cancer samples.

<p>Immunohistological staining was performed with H102 (panel A) and pT120 (panel B) antibodies on serial section of tissues. 3 and 4 are higher magnification of 1 and 2, respectively. 1 and 3 are normal prostate tissue. 5 and 6 show that pT120 β-catenin predominantly distributes to plasma membrane and nucleus in tumor samples. Bars in panels 1 and 2 equal to 50 micrometer. (C) Scatter plot for pT120 staining pattern in normal and prostate cancer samples. Normal and tumor samples were counted for the “particle” staining pattern as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033830#pone-0033830-g004" target="_blank">Fig. 4B</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033830#pone-0033830-g005" target="_blank">5B3</a>. The number on Y-axis represents the average of two independent counts. A cutoff at 30 (dashline) is used to as arbitrary definition of positive and negative staining.</p

PKD1 represses Wnt/β-catenin transcription activity.

<p>(A) PKD1 inhibits β-catenin/TCF transcription activity. Topflash assay was performed in LNCaP cells. Data were normalized by <i>Renilla</i> luciferase activity based on average of triple samples. Error bars represent standard deviation. Significant difference between the control and tested groups were determined by Student's t-test. (B) Overexpression of PKD1 in C4-2 cells results in inhibition of Wnt target genes. Representative results of semi-quantitative RT-PCR for Wnt/β-catenin target genes from two independent assays (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033830#s4" target="_blank">Materials and Methods</a>). (C) β-catenin in C4-2/PKD1 cells is co-localized with TGN marker p230 (white arrows). When the cells were treated with PKD1 small molecule inhibitor CID 755763 (50 nM) for 3days, the pattern is decreased (D). Anti β-catenin antibody H102 was used to stain endogenous β-catenin (red). TGN marker p230 and nuclei were labeled green. Images represent typical results from two tests.</p

Comparison of H102 and pT120 staining patterns.

<p>Comparison of H102 and pT120 staining patterns.</p