FIGURE 4.

AR is associated with telomeric proteins in LNCaP cells. A, telomeric proteins are overexpressed in prostate cancer cells. Whole cell lysates prepared from prostate cancer cells (LNCaP, PPC-1, and PC-3) and normal prostate epithelial cells (PrEC) were subjected to Western blot analysis to determine TRF1, TRF2, TIN2, AR, and β-actin (loading control) protein levels. B, AR and TRF2 in LNCaP cell lysates were individually immunoprecipitated (IP) using monoclonal antibodies, and immunoprecipitates and starting lysate (2% of input) were subjected to Western blot (WB) analysis of AR, TRF1, and TRF2. C, cell lysates were prepared from LNCaP cells transfected with HA-tagged TRF1 as described previously (13), and HA-TRF1 in cell lysates was immunoprecipitated using antibodies against the HA epitope. Immunoprecipitates and unprecipitated lysate (10% of input) were subjected to Western blot analysis of AR and TIN2. D, sucrose density gradient analysis of telomeric proteins and AR in LNCaP cells. Nuclear extract (NE) prepared from exponentially growing LNCaP cells was subjected to sucrose density gradient centrifugation, and the gradient was resolved into 12 fractions, which were subjected to Western blot analysis to identify the distribution of TRF1, TRF2, TIN2, TPP1, and AR in the gradient. Lamin B was used as a control. Top, top of the gradient; Bottom, bottom of the gradient; NE, Nuclear extract loaded onto the gradient.