PSMA associates with Triton X-100-DRMs upon activation.

<p>(A) LNCaP cells were activated by antibody-induced cross-linking of PSMA at 37°C or 4°C. Subsequently non activated and activated cells were solubilized with either 1% Lubrol WX or 1% Triton X-100 (w/v) in PBS. DRMs (P) and soluble (S) fractions were isolated as described in Fig. 1A. Equal amounts of total protein from all fractions were loaded for each lane on SDS gels and blotted. The distribution of PSMA activated with 7e11c mAb, canine PSMA activated with D2B mAb as well as LPH activated with 909 mAb were analyzed as negative controls. Western blots were developed using D2B mAb for PSMA and anti-GFP mAb for canine PSMA and LPH. The activation of PSMA by antibody-induced cross-linking results in redistribution of PSMA to Triton X-100-DRMs whereas the negative controls show no changes in their detergent solubility properties. P pellet, S supernatant (B) Statistical analysis of results from five independent experiments was performed using GraphPad Prism5 applying the parametric paired t test (two-tailed). Significance was accepted when p<0.05. P nAct - pellet of non activated cells, S nAct - supernatant of non activated cells, P Act - pellet of activated cells, S Act - supernatant of activated cells.