The lysates were homogenized, and the homogenates were centrifuged at 1,6006g at 4uC for 10 min

PE1 cells, the pT120 b-catenin distributes predominately as diffused cytosol protein. However, ” in a benign hyperplastic prostate epithelial BPH-1 cell line, the pT120 b-catenin shows predominate staining on plasma membrane and nucleus. The results suggest that the variation of subcellular localization of pT120 may be sensitive to cellular signaling changes in prostate cancer progression. The T120 phosphorylated b-catenin in TGN dramatically decreased in prostate cancer specimens In order to explore utility of T120 b-catenin antibody in clinical specimens immunohistochemical studies were carried out. IHC was ” performed on serial sections of paraffinized human prostate normal and cancer samples arranged on tissue arrays to compare pT120 b-catenin to total b-catenin distribution. In normal samples, the pT120 antibody typically showed diffuse cytoplasm staining with heavy TGN particles. Twenty-two of 30 benign prostate tissues demonstrated pT120 b-catenin in TGN as judged by over 30 TGN particles. In contrast, 183/197 cancer specimens showed no pT120 b-catenin accumulation in TGN compared to benign prostate tissues. Cancer specimens with Gleason grade $7 showed even lower percentage of positive TGN staining versus 14.8% positive staining from specimens with Gleason 36. T120 phosphorylated b-catenin accumulates in TGN in prostate tissue To study the pT120 b-catenin in situ, we co-stained formaldehyde-fixed, paraffin embedded benign human prostate tissue using the pT120 or H102 antibody with TGN marker p230. b-catenin visualized by conventional H102 antibody typically localizes on plasma membrane as well as TGN. In contrast, vast majority of b-catenin visualized by the pT120 antibody colocalizes with p230 on TGN. Weak staining of plasma membrane is also detected. To confirm the specificity of pT120 antibody using in immunohistochemetry, we performed peptide competition assay on pT120 antibody in IHC. The pT120 antibody staining displays a more diffuse pattern with heavy particles in cytoplasm presumably the TGN. In the presence of antigenic phosphopeptide, most staining disappeared except few spots in TGN, the most Varlitinib chemical information likely explanation for the observation is that the TGN local concentration of pT120 b-catenin is too high for the phosphopeptide to totally compete. In contrast, the non-phosphopeptide cannot compete with pT120 antibody staining in cytoplasm but not in TGN. Discussion PKD1 suppresses cancer cell epithelial to mesenchymal transition by inhibitory phosphorylation of transcription factor Snail, a known E-cadherin repressor. Overexpression of PKD1 in 5 Beta-Catenin T120 Phosphorylation C4-2 induces expression of E-cadherin and lowers tumor incidence in mice xenograft models. b-catenin serves as a co-repressor of expression of prostate cancer metastasis repressor gene KAI1 . We have previously shown that expression of KAI1 actually was induced in C4-2/PKD1 cells, although the expression of b-catenin is also upregulated. These data suggest that b-catenin protein levels do not necessarily correlate with its transcription activity. In this study, we found that the protein level of nuclear b-catenin in C-42/PKD1 is more compared to C4-2 cells, but b-catenin transcription is lower in C4-2/PKD1 cells. This finding cannot be merely explained by induction of E-cadherin expression, because Ecadherin is thought to sequester cytoplasm b-catenin pool and decrease availability of free b-catenin for transcription in nucleus. The pT120 antibody unveils